PKLR mutations in pyruvate kinase deficient Polish patients: Functional characteristics of c.101-1G > A and c.1058delAAG variants.

Pyruvate kinase (PK) deficiency is a rare autosomal recessive disorder characterized by chronic hemolytic anemia of variable severity. Nine Polish patients with severe hemolytic anemia but normal PK activity were found to carry mutations in the PKLR gene encoding PK, five already known ones and one novel (c.178C > T). We characterized two of the known variants by molecular modeling (c.1058delAAG) and minigene splicing analysis (c.101-1G > A). The former gives a partially destabilized PK tetramer, likely of suboptimal activity, and the c.101-1G > A variant gives alternatively spliced mRNA carrying a premature stop codon, encoding a severely truncated PK and likely undergoing nonsense-mediated decay.

Hereditary xerocytosis - spectrum and clinical manifestations of variants in the PIEZO1 gene, including co-occurrence with a novel ß-globin mutation.

Hereditary xerocytosis (HX) is a rare, autosomal dominant congenital hemolytic anemia (CHA) characterized by erythrocyte dehydration with presentation of various degrees of hemolytic anemia. HX is often misdiagnosed as hereditary spherocytosis or other CHA. Here we report three cases of suspected HX and one case of HX associated with ß-thalassemia. Sanger method was used for sequencing cDNA of the PIEZO1 gene. Variants were evaluated for potential pathogenicity by MutationTaster, PROVEAN, PolyPhen-2 and M-CAP software, and by molecular modeling. Four different variants in the PIEZO1 gene were found, including three substitutions (p.D669H, p.D1566G, p.T1732 M) and one deletion (p.745delQ). In addition, in the patient with the p.T1732 M variant we detected a 12-nucleotide deletion in the ß-globin gene leading to a deletion of amino acids 62AHGK65. The joint presence of mutations in two different genes connected with erythrocytes markedly aggravated the presentation of the disease. Bioinformatic analysis and molecular modeling strongly indicated likely deleterious effects of all four PIEZO1 variants, but co-segregation analysis showed that the p.D1566G substitution is in fact non-pathogenic. Identification of causative mutations should improve the diagnosis and management of HX and provide a new insight into the molecular basis of this complex red blood cell abnormality.

Graphene oxide down-regulates genes of the oxidative phosphorylation complexes in a glioblastoma.

BACKGROUND: Recently different forms of nanographene were proposed as the material with high anticancer potential. However, the mechanism of the suppressive activity of the graphene on cancer development remains unclear. We examined the effect of oxygenated, reduced and pristine graphene on the gene expression in glioblastoma U87 cell line. RESULTS: Conducting microarrays and RT-qPCR analysis we explored that graphene oxide (rather than reduced graphene oxide and pristine graphene) down-regulates the mRNA expression of mitochondrial oxidative phosphorylation (OXPHOS) nuclear genes of complexes I, III, IV and V. The presented results provide first evidence for the hypothesis that the suppressed growth of GBM can be the consequence of down-regulation of OXPHOS protein expression and decreased ATP level. CONCLUSIONS: We suggest that changes in the expression of OXPHOS genes identified in our study may mediate the anti-proliferative and anti-migratory effects of graphene oxide in glioblastoma cells. However, further investigations with different cell lines, regarding expression, regulation and activity of OXPHOS genes identified in our study is necessary to elucidate the mechanism mediating the anti-proliferative and anti-migratory effects of graphene oxide in glioblastoma cells.

Two novel C-terminal frameshift mutations in the ß-globin gene lead to rapid mRNA decay.

BACKGROUND: The thalassemia syndromes are classified according to the globin chain or chains whose production is affected. ß-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the ß-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level. METHODS: Two cases of Polish patients with hereditary hemolytic anemia suspected of thalassemia were studied. DNA sequencing and mRNA quantification were performed. Stable human cell lines which express wild-type HBB and mutated versions were used to verify that detected mutation are responsible for mRNA degradation. RESULTS: We identified two different frameshift mutations positioned in the third exon of HBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although detected mutations are not expected to induce NMD, the mutant version of mRNA is not detectable. Restoring of the open reading frame brought back the RNA to that of the wild-type level. CONCLUSION: Our results show that a lack of natural stop codon due to the frameshift in exon 3 of ß-globin gene causes rapid degradation of its mRNA and indicate existence of novel surveillance pathway.

Feto-maternal haemorrhage assessment in a woman with a large population of red blood cells containing fetal haemoglobin.

BACKGROUND: FMH quantification is necessary to calculate an individual dose of prophylactic anti-RhD immunoglobulin and to diagnose fetal anaemia causes. We encountered a healthy woman with a numerous RBCs containing fetal haemoglobin (HbF). AIMS: To investigate the cause of this sign and the correct evaluation of fetal RBCs in maternal circulation. MATERIALS AND METHODS: Patients samples and artificial mixtures were tested by microscopic Kleihaur-Betke (KB) and flow cytometric (FC) tests with anti-HbF + anti-CA (carbonic anhydrase), and with anti-D. The patient's blood count with reticulocyte parameters, and concentration of bilirubin, haptoglobin, iron, transferrin, ferritin, hepcidin, sTR, HbF, HbA2 were measured. Genes coding the beta- and gamma-globin were sequenced. RESULTS: It was impossible to distinguish the population of fetal and maternal HbF positive cells using KBT and FC with anti-HbF. Application of anti-CA and anti-D allowed to separate them. Maternal blood haematological and biochemical parameters were normal but HbF was 3.3% of total Hb concentration (normal < 1%). There were no mutations in the beta- and gamma-globin genes, but Xmn I polymorphism at -158 position in gamma-globin gene was detected in the homozygous state. CONCLUSION: A very large population of HbF positive cells sometimes can be detect in a healthy woman. Implementation of the various procedures for FMH assessment is necessary in the such case, otherwise, the detection of fetal erythrocytes may not be possible or can give false results.

The effects of statins on the mevalonic acid pathway in recombinant yeast strains expressing human HMG-CoA reductase.

BACKGROUND: The yeast Saccharomyces cerevisiae can be a useful model for studying cellular mechanisms related to sterol synthesis in humans due to the high similarity of the mevalonate pathway between these organisms. This metabolic pathway plays a key role in multiple cellular processes by synthesizing sterol and nonsterol isoprenoids. Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the cholesterol synthesis pathway. However, the effects of statins extend beyond their cholesterol-lowering action, since inhibition of HMGR decreases the synthesis of all products downstream in the mevalonate pathway. Using transgenic yeast expressing human HMGR or either yeast HMGR isoenzyme we studied the effects of simvastatin, atorvastatin, fluvastatin and rosuvastatin on the cell metabolism. RESULTS: Statins decreased sterol pools, prominently reducing sterol precursors content while only moderately lowering ergosterol level. Expression of genes encoding enzymes involved in sterol biosynthesis was induced, while genes from nonsterol isoprenoid pathways, such as coenzyme Q and dolichol biosynthesis or protein prenylation, were diversely affected by statin treatment. Statins increased the level of human HMGR protein substantially and only slightly affected the levels of Rer2 and Coq3 proteins involved in non-sterol isoprenoid biosynthesis. CONCLUSION: Statins influence the sterol pool, gene expression and protein levels of enzymes from the sterol and nonsterol isoprenoid biosynthesis branches and this effect depends on the type of statin administered. Our model system is a cheap and convenient tool for characterizing individual statins or screening for novel ones, and could also be helpful in individualized selection of the most efficient HMGR inhibitors leading to the best response and minimizing serious side effects.

Will global transcriptome analysis allow the detection of novel prognostic markers in coronary artery disease and heart failure?

Coronary artery disease (CAD) is one of the leading causes of death in the developed countries. Myocardial infarction (MI) is an acute episode of CAD that results in myocardial injury and subsequent heart failure (HF). In the acute phase of MI several risk factors for future cardiovascular events have been found. The molecular mechanisms of these disorders are still unknown, but altered gene expression may play an important role in the development and progression of cardiovascular diseases. High-throughput techniques should greatly facilitate the elucidation of the mechanisms and provide novel insights into the pathophysiology of cardiovascular diseases. In this review we focus on the perspectives of gene-expression profiling conducted on cardiac tissues and blood for the determination of novel diagnostic and prognostic markers and therapeutic targets.

Hypothalamic Neurochemical Changes in Long-Term Recovered Bilateral Subdiaphragmatic Vagotomized Rats.

Background: Vagus nerve is one of the crucial routes in communication between the immune and central nervous systems. The impaired vagal nerve function may intensify peripheral inflammatory processes. This effect subsides along with prolonged recovery after permanent nerve injury. One of the results of such compensation is a normalized plasma concentration of stress hormone corticosterone - a marker of hypothalamic-pituitary-adrenal (HPA) axis activity. In this work, we strive to explain this corticosterone normalization by studying the mechanisms responsible for compensation-related neurochemical alterations in the hypothalamus. Materials and Methods: Using microarrays and high performance liquid chromatography (HPLC), we measured genome-wide gene expression and major amino acid neurotransmitters content in the hypothalamus of bilaterally vagotomized rats, 1 month after surgery. Results: Our results show that, in the long term, vagotomy affects hypothalamic amino acids concentration but not mRNA expression of tested genes. Discussion: We propose an alternative pathway of immune to CNS communication after vagotomy, leading to activation of the HPA axis, by influencing central amino acids and subsequent monoaminergic neurotransmission.

Expression of versican mRNA transcript to predict cardiac remodelling after myocardial infarction.

BACKGROUND: Adverse left-ventricular remodelling (LVR) is defined as an increase in end-diastolic left-ventricular volume by 20% 6 months after acute myocardial infarction (AMI). LVR is associated with cardiac dysfunction, therefore deteriorating the prognosis. AIMS: We aimed to compare the concentrations of messenger RNA transcripts in the peripheral blood of patients with and without LVR at 6 months. METHODS: The study included 75 patients with first ST-elevation myocardial infarction (STEMI) treated with percutaneous coronary intervention. Whole blood concentrations of 6 transcripts were determined 24 hours after AMI using droplet digital polymerase chain reaction. The correlations between mRNA transcript expression and left ventricular ejection fraction (LVEF) and N-terminal-pro B type natriuretic peptide (NT-proBNP) concentration were evaluated. RESULTS: Among 75 patients, 4 were lost to follow-up and 71 were included in the analysis. Seventeen (24%) patients developed LVR at 6 months. Versican (VCAN) mRNA expression was lower in patients who developed LVR, compared to those who did not (P = 0.02), and discriminated between these patients (area under the ROC curve 67%; P = 0.04). Expression of VCAN transcript < 75.3 normalized units predicted LVR with 71% sensitivity and 67% specificity. In a multivariable regression analysis, VCAN expression remained the only independent predictor of LVR (OR 3.475; 95% CI, 1.000-12.075; P = 0.04). CONCLUSIONS: Dysregulation of VCAN expression in the acute phase of AMI may contribute to LVR at 6 months. Whether decreased expression of VCAN might be a useful tool to predict LVR in clinical practice remains to be established.

A Family Affected by a Life-Threatening Erythrocyte Defect Caused by Pyruvate Kinase Deficiency With Normal Iron Status: A Case Report.

BACKGROUND: Red cell pyruvate kinase deficiency (PKD) is a defect of glycolysis causing congenital non-spherocytic hemolytic anemia. PKD is transmitted as an autosomal recessive trait. The clinical features of PKD are highly variable, from mild to life-threatening anemia which can lead to death in the neonatal period. Most patients with PKD must receive regular transfusions in early childhood and as a consequence suffer from iron overloading. PATIENT: Here, we report a Polish family with life-threatening hemolytic anemia of unknown etiology. Whole exome sequencing identified two heterozygous mutations, c.1529 G > A (p.R510Q) and c.1495 T > C (p.S499P) in the PKLR gene. Molecular modeling showed that the both PKLR mutations are responsible for major disturbance of the protein structure and functioning. Despite frequent transfusions the patients do not show any signs of iron overload and hepcidin, a major regulator of iron uptake, is undetectable in their serum. The patients were homozygous for the rs855791 variant of the TMPRSS6 gene which has earlier been shown to down-regulate iron absorption and accumulation. CONCLUSION: The lack of iron overload despite a reduced level of hepcidin in two transfusion-dependent PKD patients suggests the existence of a hepcidin-independent mechanism of iron regulation preventing iron overloading.

Changes in MicroRNA Expression during Rabbit Hemorrhagic Disease Virus (RHDV) Infection.

Current knowledge on the role of microRNAs (miRNAs) in rabbit hemorrhagic disease virus (RHDV) infection and the pathogenesis of rabbit hemorrhagic disease (RHD) is still limited. RHDV replicates in the liver, causing hepatic necrosis and liver failure. MiRNAs are a class of short RNA molecules, and their expression profiles vary over the course of diseases, both in the tissue environment and in the bloodstream. This paper evaluates the expression of miRNAs in the liver tissue (ocu-miR-122-5p, ocu-miR-155-5p, and ocu-miR-16b-5p) and serum (ocu-miR-122-5p) of rabbits experimentally infected with RHDV. The expression levels of ocu-miR-122-5p, ocu-miR-155-5p, and ocu-miR-16b-5p in liver tissue were determined using reverse transcription quantitative real-time PCR (RT-qPCR), and the expression level of circulating ocu-miR-122-5p was established using droplet digital PCR (ddPCR). The expression levels of ocu-miR-155-5p and ocu-miR-16b-5p were significantly higher in the infected rabbits compared to the healthy rabbits (a fold-change of 5.8 and 2.5, respectively). The expression of ocu-miR-122-5p was not significantly different in the liver tissue from the infected rabbits compared to the healthy rabbits (p = 0.990), while the absolute expression level of the circulating ocu-miR-122-5p was significantly higher in the infected rabbits than in the healthy rabbits (p < 0.0001). Furthermore, a functional analysis showed that ocu-miR-155-5p, ocu-miR-16b-5p, and ocu-miR-122-5p can regulate the expression of genes involved in processes correlated with acute liver failure (ALF) in rabbits. Search tool for the retrieval of interacting genes/proteins (STRING) analysis showed that the potential target genes of the three selected miRNAs may interact with each other in different pathways. The results indicate the roles of these miRNAs in RHDV infection and over the course of RHD and may reflect hepatic inflammation and impairment/dysfunction in RHD.

Serum microRNA in patients undergoing atrial fibrillation ablation.

MicroRNAs mediate posttranscriptional gene regulation. The aim of the study was to find a microRNA predictor of successful atrial fibrillation (AF) ablation. A total of 109 patients undergoing first-time AF ablation were included. Nineteen patients were selected to undergo serum microRNA sequencing (study group). The sequencing data were used to select several microRNAs that correlated with 12-month recurrences after AF ablation. Those microRNAs were validated by digital droplet PCR in samples from remaining 90 patients. All patients underwent pulmonary vein isolation (RF ablation, contact force catheter, electroanatomical system). The endpoint of the study was the 12-month AF recurrence rate; the overall recurrence rate was 42.5%. In total, levels of 34 miRNAs were significantly different in sera from patients with AF recurrence compared to patients without AF recurrence. Six microRNAs (miR-183-5p, miR-182-5p, miR-32-5p, miR-107, miR-574-3p, and miR-144-3p) were validated in the whole group. Data from the validation group did not confirm the observations from the study group, as no significant differences were found between miRNAs serum levels in patients with and without recurrences 12 months after AF ablation.

Novel beta-spectrin mutations in hereditary spherocytosis associated with decreased levels of mRNA.

Hereditary spherocytosis (HS) is one of the most frequent and heterogeneous inherited haemolytic anaemias. It is associated with abnormalities of several erythrocyte membrane proteins. We investigated relative mRNA quantification of red blood cell membrane protein genes using real-time quantitative polymerase chain reaction (qPCR) in order to better characterize HS cases and to select genes to search for mutations in patients with spherocytosis. qPCR experiments indicated that the spectrin beta gene (SPTB) could be involved in anaemia pathogenesis. DNA analysis of SPTB in the HS subjects with decreased SPTB mRNA levels revealed the presence of five previously undescribed mutations: R1756X, 781delT and IVS22nt-4G>A, 1502insA and IVS20nt-2A>G.

The use of real-time PCR technique in the detection of novel protein 4.2 gene mutations that coexist with thalassaemia alpha in a single patient.

Alpha-thalassaemia is a very rare disease in Northern Europe in contrast to hereditary spherocytosis that is associated with red blood cell membrane defects. We report here alpha-thalassaemia case who was also found to bear the erythrocyte membrane protein 4.2 gene mutations. mRNA relative quantification of red cell membrane protein genes in a Polish patient with alpha-thalassaemia trait indicated EPB42 as the gene that could also be involved in anaemia pathogenesis. Sequencing revealed the presence of two novel mutations in the protein 4.2 gene: a G1701A genetic change that predicts an alanine to threonine at position 567 of the protein (A567T) and a T-->A substitution that is located at position +6 of the donor splice site of intron 2 (IVS2nt+6T>A). This is the sixth variant of the erythrocyte membrane protein 4.2 gene mutations identified outside the Japanese population.

Theoretical model of thalassemic erythrocyte shape transformation.

Our earlier model of reticulocyte shape transformation [Pawlowski, P.H., Burzynska, B., Zielenkiewicz, P., 2006. Theoretical model of reticulocyte to erythrocyte shape transformation. J. Theor. Biol. 243, 24-38] was applied to explain the morphological properties of thalassemic erythrocytes. Modification of the standard set of parameters of the model, describing minimal cell volume, membrane bending rigidity, and membrane tension, allowed for simulation of development of alpha- and beta-thalassemic cells from splenectomized and nonsplenectomized individuals. This resulted in observation of thin rim discocytes, tailed erythrocytes and oval forms, as well as in differentiation of time of the cell shape metamorphosis. A comparative analysis of the susceptibility of thalassemic and normal erythrocytes to undergo deformation as well of their stability was performed.