Evaluation of an automatic HPLC analyser for thalassemia and haemoglobin variants screening

In this paper the authors report the evolution of a new automatic HPLC analyser for screening haemoglobinopathies. HbA2 and F determinations are accurate and reproducible. The analysis time is short (6.5 min) and there is a good separation between the HbA2 values of β-thalassemia carriers from normals and α-thalassemia carriers, with no overlap between these groups. In addition, the system is also able to detect and quantitate most of the haemoglobin variants, particularly those (HbS, HbC, HbE and Hb Lepore) able to interact with β-thalassemia and could make haemoglobin electrophoresis unnecessary in all samples. The ease of operation and the limited technical work make this system especially suitable for laboratories with a high workload and allow the cost of screening to be reduced

Deficiency in interferon type 1 receptor improves definitive erythropoiesis in Klf1 null mice

A key regulatory gene in definitive erythropoiesis is the transcription factor Krüppel-like factor 1 (Klf1). Klf1 null mice die in utero by day 15.5 (E15.5) due to impaired definitive erythropoiesis and severe anemia. Definitive erythropoiesis takes place in erythroblastic islands in mammals. Erythroblastic islands are formed by a central macrophage (Central Macrophage of Erythroblastic Island, CMEI) surrounded by maturating erythroblasts. Interferon-β (IFN-β) is activated in the fetal liver’s CMEI of Klf1 null mice. The inhibitory effect of IFN-β on erythropoiesis is known and, therefore, we speculated that IFN-β could have contributed to the impairment of definitive erythropoiesis in Klf1 knockout (KO) mice fetal liver. To validate this hypothesis, in this work we determined whether the inactivation of type I interferon receptor (Ifnar1) would ameliorate the phenotype of Klf1 KO mice by improving the lethal anemia. Our results show a prolonged survival of Klf1/Ifnar1 double KO embryos, with an improvement of the definitive erythropoiesis and erythroblast enucleation, together with a longer lifespan of CMEI in the fetal liver and also a restoration of the apoptotic program. Our data indicate that the cytotoxic effect of IFN-β activation in CMEI contribute to the impairment of definitive erythropoiesis associated with Klf1 deprivation

IFNL3 polymorphisms and HCV infection in patients with beta thalassemia

Background and relationale for the study. Genome-wide association studies have identified host genetic variation to be critical for spontaneous clearance and treatment response in patients infected with hepatitis C virus. Recently, the role of the IFNL3 polymorphisms in influencing the spontaneous clearance of HCV, the response to interferon and the progression of liver fibrosis, was also demonstrated in patients with thalassemia major infected by genotype 1b. In the present study we retrospectively analyzed 368 anti-HCV positive patients with beta-thalassemia at two Italian major centers in Cagliari and Torino. Results. C/C variant of polymorphism rs12979860 was related to response to interferon treatment and, above all, to spontaneous clearance of the virus. However, the positive predictive power was stronger for viral persistence than spontaneous clearance and in such respect the TT allele was more predictive than CC. The methylation associated polymorphism rs4803221 had independent effects with respect to rs12979860 and the haplotype tagged by SNP rs12979860 and rs4803221 significantly could improve the viral clearance prediction in infected patients. Neither necroinflammation or bilirubin values in the chronic phase of the hepatitis C were related to IFNL3 polymorphisms. No relation among IFNL3 polymorphisms and fibrosis stage directly shown by the liver biopsy was found. Conclusions. Also in thalassemia the SNPs on chromosome 19q13 closely associates with spontaneous and treatment-induced HCV clearance. The viral clearance prediction is significantly improved by the haplotype tagged by SNP rs12979860 and rs4803221. Neither necroinflammation, bilirubin values or fibrosis stage seem to be related to IFNL3 polymorphisms

Delta-Globin Gene Expression Is Enhanced in vivo by Interferon Type I

Beta hemoglobinopathies are widely spread monogenic lethal diseases. Delta-globin gene activation has been proposed as a possible approach for curing these pathologies. The therapeutic potential of delta-globin, the non-alpha component of Hemoglobin A2 (α2δ2; HbA2), has been demonstrated in a mouse model of beta thalassemia, while its anti-sickling effect, comparable to that of gamma globin, was established some time ago. Here we show that the delta-globin mRNA level is considerably increased in a Deoxyribonuclease II-alpha knockout mouse model in which type 1 interferon (interferon beta, IFNb) is activated. IFNb activation in the fetal liver improves the delta-globin mRNA level, while the beta-globin mRNA level is significantly reduced. In addition, we show that HbA2 is significantly increased in patients with multiple sclerosis under type 1 interferon treatment. Our results represent a proof of principle that delta-globin expression can be enhanced through the use of molecules. This observation is potentially interesting in view of a pharmacological approach able to increase the HbA2 level

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases

Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR)

© 2016 The Author(s). Objectives: Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. Methods: The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. Results: A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. Conclusion: We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts

A Biochip reader for qualitative and quantitative analysis of images, in particular for the analysis of single or multiple Biochips

A biochip reader for qualitative and quantitative analysis of images, in particular for the analysis of single or multiple biochips with different colorimetric signals for different targets of biological interest such as drugs or nucleic acids, fat acids and proteins from viruses, prokaryotes and eukaryotes organisms, obtained from human, animal, vegetal or environmental biological samples. Said biochip reader comprises: - an optical head 18, able to moving itself in one direction Y, comprising: - at least one visible light source 20 or 21 - at least two CCD sensors 16 and 17, attached each other; each CCD comprising a reflective lens set 24 and a focalising lens 25; - control means, able to control said optical head 18 and its one-dimensional motion. Being each CCDs attached each other, then each half, or portion of, image has an identical position or angle error of the other half, or portion of, image, so as the joining process becomes immediat