Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR

Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation

Genotyping Of Kell, Duffy, Kidd And Rhd In Patients With β Thalassemia

Determination of Rh, Kell, Duffy and Kidd phenotypes in addition to ABO is used to prevent the alloimmunization to red blood cells (RBCs) antigens and as part of the antibody identification process in patients with β Thalassemia. However, phenotyping in these patients can be time consuming and difficult to interpret. In these situations, it would be valuable to have an alternative to hemagglutination tests to determine the patient's antigen profile. We used PCR-RFLP to genotype such patients. DNA was prepared from 50 patients with β Thalassemia who had been phenotyped by routine hemagglutination, and tested for Kell, Kidd, Duffy/ GATA mutation by PCR-RFLP. RHD/non-D was analysed by PCR product size associated to RHD gene sequence in intron 4 and exon 10/3'UTR. The genotyping assays were performed without knowledge of phenotype results. For RHD/non-D, 47 were RhD+ and RHD+/RHCE+, and 3 were RhD- and RHD-/RHCE+. For Kell, 48 kk were K2K2 and 2 Kk were K1K2. For Duffy, of 44 samples that had normal GATA box, 8 Fy(a+b-) were FYA/FYA, 15 Fy(a+b+) were FYB/FYB, and 19 Fy(a+b+) were FYA/FYB; of the other 4 samples 3 were FYA/FYB and heterozygous GATA mutation, and 1 Fy(a-b-) was FYB/FYB, homozygous GATA mutation. Two samples phenotyped as Fy(a+b-) that had normal GATA, presented the 265T/298 A mutations and two samples phenotyped as Fy(a-b+) were genotyped was FYA/FYB. For Kidd, 15 Jk(a+b) were JKA/JKA, 12 Jk(a-b+) were JKB/JKB, and 20 Jk(a+b+) were JKA/JKB. Three samples phenotyped as JK(a+b+) were genotyped as JKB/JKB. Genotype is more accurate than phenotype for determination of blood groups in polytransfused patients with β Thalassemia. Genotyping in these patients can be helpful to select antigen-negative RBCs for transfusion.2226976Blumberg, N., Peck, K., Ross, K., Avila, E., Immune response to chronic red blood cell transfusion (1983) Vox Sang, 44, pp. 212-217Economidou, J., Constantoulakis, M., Augoustaki, O., Adinolfi, M., Frequency of antibodies to various antigenic determinants in polytransfused patients with homozygous thalassemia in Greece (1971) Vox Sang, 20, p. 252Sirchia, G., Zanella, A., Parravicini, A., Morelati, F., Rebulla, P., Masera, G., Red cell alloantibodies in patients with thalassemia major. Results of na Italian cooperative study (1985) Transfusion, 25, p. 110Spanos, T., Karageorga, M., Ladis, V., Peristeri, J., Hatziliami, A., Kattamis, C., Red cell alloantibodies in patients with thalassemia (1990) Vox Sang, 58, p. 50Greenwalt, T.J., Zelenski, K.R., Transfusion support for hemoglobinopathies (1984) Clin. Haematol., 13, pp. 151-165Charache, S., Problems in transfusion therapy (1990) N. Engl. J. Med., 322, pp. 1666-1668. , editorialPerkins, H.A., The safety of the blood supply: Making decisions in transfusion medicine (1992) Blood Safety: Current Challenges, pp. 125-150. , Nance SJ, ed. Bethesda: American Association of Blood BanksColes, S.M., Klein, H.G., Holland, P.V., Alloimmunization in two multitransfused patient populations (1981) Transfusion, 21, pp. 462-466Michail-Merianou, V., Pamphili-Panouspoulou, L., Piperi-Lowes, L., Pelegrinis, E., Karaklis, A., Alloimmunization to red cell antigens in thalassemia: Comparative study of usual versus better-match transfusion programmes (1987) Vox Sang, 52, p. 95Reid, M.E., Yazdanbakhsh, K., Molecular insights into blood groups and implications for blood transfusions (1998) Current Opinion in Hematology, 5, pp. 93-102Avent, N.D., Human erythrocyte antigen expression: Its molecular bases (1997) Br. J. Biom. Sci., 54, pp. 16-37Lee, T.H., Donegan, E., Slichter, S., Bush, M.P., Transient increase in circulating donor leucocytes after allogeneic transfusions in Immunocompetent recipients compatible with donor cell proliferation (1995) Blood, 85, pp. 1207-1214Adams, F.T., Davenport, R.D., Rcardon, D.A., Roth, M.S., Detection of circulating donor white blood cells in patients receiving multiple trasnfusions (1992) Blood, 80, pp. 551-555Lee, T.-H., Paglieroni, T., Ohro, H., Holland, P.V., Bush, M.P., Longterm multi-lineage chimerism of donor leucocytes in transfused trauma patients (1996) Blood, 88, p. 265. , abstrRios, M., Cash, K., Strupp, A., Uehlinger, J., Reid, M.E., DNA from urine sediment or buccal cells can be used for blood group molecular genotyping (1999) Immunehematology, 15, pp. 61-65Reid, M.E., Rios, M., Powell, D., Charles-Pierre, D., Malavade, V., DNA from blood samples can be used to genotype patients who have recently received a transfusion (2000) Transfusion, 40, pp. 1-6Davies, L., Dibner, M.D., Battey, J.F., (1986) Basic Methods in Molecular Biology, , Elsevier Science Publishing Co. Inc., New YorkLee, S., Wu, X., Reid, M.E., Zelinski, T., Redman, C., Molecular basis of the Kell (K1) phenotype (1995) Blood, 85, pp. 912-916Olivès, B., Merriman, M., Bailly, P., Bain, S., Barnett, A., Todd, J., Cartron, J.-P., Merriman, T., The molecular basis of the Kidd blood group polymorphism and its lack of association with type 1 diabetes susceptibility (1997) Hum. Mol. Genet., 6, pp. 1017-1020Chaudhuri, A., Polyakova, J., Zbrezezna, V., Williams, K., Gulati, S., Pogo, A.O., Cloning of glycoprotein D cDNA, which encodes the major subunit of the Duffy blood group system and the receptor for the Plasmodium vivax malaria parasite (1993) Proc. Natl. Acad. Sci. USA, 90, pp. 10793-10797Iwamoto, S., Omi, T., Kajii, E., Ikemoto, S., Genomic organization of the glycophorin D gene: Duffy blood group Fy a/Fy b alloantigen system is associated with a polymorphism at the 44-amino residue (1995) Blood, 85, pp. 622-626Tournamille, C., Collin, Y., Cartron, J.-P., Van Le Kim, C., Disruption of a GATA motif in the Duffy gene promotor abolishes erythroid gene expression in Duffy-negative individuals (1995) Nature Genet., 10, pp. 224-228Rios, M., Reid, M.E., Naime, D., Chaudhuri, A., Pogo, A.O., Bianco, C., Importance of GATA box analysis in genotyping for the Duffy blood group system (1997) Transfusion, 37 (S), pp. 101S. , abstrZimmerman, P.A., Woolley, I., Masinde, G.L., Miller, S.M., McNamara, D.T., Hazlett, F., Mgone Alpers, M.P., Kazura, J.W., Emergence of FY*A(null) in a Plasmodium vivax-endemic region of Papua New Guinea (1999) Proc Natl Acad Sci. USA, 96 (24), pp. 13973-13977. , Nov 23Olsson, M.L., Hansson, C., Akesson, I.E., Avent, N.D., Daniels, G.L., Detection of the common alleles at the Duffy blood group locus by allele-specific primer PCR (1997) Transfusion, 37 (S), pp. 102S. , abstrCartron, J.-P., Bailly, P., Van Le Kim, C., Insights into the structure and function of membrane polypeptides carrying blood group antigens (1998) Vox Sang, 74 (SUPPL. 2), pp. 29-64Huang, C.H., Molecular insights into the Rh protein family and associated antigens (1997) Curr Opin Hematol., 4, pp. 94-103Huang, C.H., Blumenfeld, O.O., MNSs blood groups and major glycophorins: Molecular basis for allelic variation (1995) Molecular Basis of Major Human Blood Group Antigens, pp. 153-183. , cartron J-P, Pouger P, eds. New York: Plenum PressAvent, N.D., Reid, M.E., The Rh Blood group system: A review (2000) Blood, 95, pp. 1-1

Search for vector-boson resonances decaying to a top quark and bottom quark in the lepton plus jets final state in pp collisions at s=13 TeV with the ATLAS detector

A search for new charged massive gauge bosons, W, is performed with the ATLAS detector at the LHC. Data were collected in proton–proton collisions at a center-of-mass energy of s=13 TeV and correspond to an integrated luminosity of 36.1 fb. This analysis searches for W bosons in the W→tb¯ decay channel in final states with an electron or muon plus jets. The search covers resonance masses between 0.5 and 5.0 TeV and considers right-handed W bosons. No significant deviation from the Standard Model (SM) expectation is observed and upper limits are set on the W→tb¯ cross section times branching ratio and the W boson effective couplings as a function of the W boson mass. For right-handed W bosons with coupling to the SM particles equal to the SM weak coupling constant, masses below 3.15 TeV are excluded at the 95% confidence level. This search is also combined with a previously published ATLAS result for W→tb¯ in the fully hadronic final state. Using the combined searches, right-handed W bosons with masses below 3.25 TeV are excluded at the 95% confidence level.Peer Reviewe

Search for heavy particles decaying into a top-quark pair in the fully hadronic final state in pp collisions at s =13 TeV with the ATLAS detector

A search for new particles decaying into a pair of top quarks is performed using proton-proton collision data recorded with the ATLAS detector at the Large Hadron Collider at a center-of-mass energy of s=13 TeV corresponding to an integrated luminosity of 36.1 fb-1. Events consistent with top-quark pair production and the fully hadronic decay mode of the top quarks are selected by requiring multiple high transverse momentum jets including those containing b-hadrons. Two analysis techniques, exploiting dedicated top-quark pair reconstruction in different kinematic regimes, are used to optimize the search sensitivity to new hypothetical particles over a wide mass range. The invariant mass distribution of the two reconstructed top-quark candidates is examined for resonant production of new particles with various spins and decay widths. No significant deviation from the Standard Model prediction is observed and limits are set on the production cross-section times branching fraction for new hypothetical Z′ bosons, dark-matter mediators, Kaluza-Klein gravitons and Kaluza-Klein gluons. By comparing with the predicted production cross sections, the Z′ boson in the topcolor-assisted-technicolor model is excluded for masses up to 3.1-3.6 TeV, the dark-matter mediators in a simplified framework are excluded in the mass ranges from 0.8 to 0.9 TeV and from 2.0 to 2.2 TeV, and the Kaluza-Klein gluon is excluded for masses up to 3.4 TeV, depending on the decay widths of the particles.Peer Reviewe

Search for pairs of highly collimated photon-jets in pp collisions at s =13 TeV with the ATLAS detector

Results of a search for the pair production of photon-jets - collimated groupings of photons - in the ATLAS detector at the Large Hadron Collider are reported. Highly collimated photon-jets can arise from the decay of new, highly boosted particles that can decay to multiple photons collimated enough to be identified in the electromagnetic calorimeter as a single, photonlike energy cluster. Data from proton-proton collisions at a center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 36.7 fb-1, were collected in 2015 and 2016. Candidate photon-jet pair production events are selected from those containing two reconstructed photons using a set of identification criteria much less stringent than that typically used for the selection of photons, with additional criteria applied to provide improved sensitivity to photon-jets. Narrow excesses in the reconstructed diphoton mass spectra are searched for. The observed mass spectra are consistent with the Standard Model background expectation. The results are interpreted in the context of a model containing a new, high-mass scalar particle with narrow width, X, that decays into pairs of photon-jets via new, light particles, a. Upper limits are placed on the cross section times the product of branching ratios σ×B(X→aa)×B(a→γγ)2 for 200 GeV<mX<2 TeV and for ranges of ma from a lower mass of 100 MeV up to between 2 and 10 GeV, depending upon mX. Upper limits are also placed on σ×B(X→aa)×B(a→3π0)2 for the same range of mX and for ranges of ma from a lower mass of 500 MeV up to between 2 and 10 GeV.Peer Reviewe

Search for tt¯ resonances in fully hadronic final states in pp collisions at √s = 13 TeV with the ATLAS detector

This paper presents a search for new heavy particles decaying into a pair of top quarks using 139 fb of proton-proton collision data recorded at a centre-of-mass energy of s = 13 TeV with the ATLAS detector at the Large Hadron Collider. The search is performed using events consistent with pair production of high-transverse-momentum top quarks and their subsequent decays into the fully hadronic final states. The analysis is optimized for resonances decaying into a tt¯ pair with mass above 1.4 TeV, exploiting a dedicated multivariate technique with jet substructure to identify hadronically decaying top quarks using large-radius jets and evaluating the background expectation from data. No significant deviation from the background prediction is observed. Limits are set on the production cross-section times branching fraction for the new Z′ boson in a topcolor-assisted-technicolor model. The Z′ boson masses below 3.9 and 4.7 TeV are excluded at 95% confidence level for the decay widths of 1% and 3%, respectively. [Figure not available: see fulltext.

Measurement of W±Z production cross sections and gauge boson polarisation in pp collisions at √s=13TeV with the ATLAS detector

This paper presents measurements of WZ production cross sections in pp collisions at a centre-of-mass energy of 13 TeV. The data were collected in 2015 and 2016 by the ATLAS experiment at the Large Hadron Collider, and correspond to an integrated luminosity of 36.1fb-1. The WZ candidate events are reconstructed using leptonic decay modes of the gauge bosons into electrons and muons. The measured inclusive cross section in the detector fiducial region for a single leptonic decay mode is σW±Z→ℓ′νℓℓfid.=63.7±1.0(stat.)±2.3(syst.)±1.4(lumi.) fb, reproduced by the next-to-next-to-leading-order Standard Model prediction of 61.5-1.3+1.4 fb. Cross sections for WZ and WZ production and their ratio are presented as well as differential cross sections for several kinematic observables. An analysis of angular distributions of leptons from decays of W and Z bosons is performed for the first time in pair-produced events in hadronic collisions, and integrated helicity fractions in the detector fiducial region are measured for the W and Z bosons separately. Of particular interest, the longitudinal helicity fraction of pair-produced vector bosons is also measured.We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Australia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DRF/IRFU, France; SRNSFG, Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland; FCT, Portugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Federation; JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, UK; DOE and NSF, USA. In addition, individual groups and members have received support from BCKDF, CANARIE, CRC and Compute Canada, Canada; COST, ERC, ERDF, Horizon 2020, and Marie Skłodowska-Curie Actions, European Union; Investissements d’ Avenir Labex and Idex, ANR, France; DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia programmes co-financed by EU-ESF and the Greek NSRF, Greece; BSF-NSF and GIF, Israel; CERCA Programme Generalitat de Catalunya, Spain; The Royal Society and Leverhulme Trust, UK. The crucial computing support from all WLCG partners is acknowledged gratefully, in particular from CERN, the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF (Denmark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (UK) and BNL (USA), the Tier-2 facilities worldwide and large non-WLCG resource providers. Major contributors of computing resources are listed in Ref. [106].Peer Reviewe

Measurement of the t t Z and t t W cross sections in proton-proton collisions at s =13 TeV with the ATLAS detector

A measurement of the associated production of a top-quark pair (tt) with a vector boson (W, Z) in proton-proton collisions at a center-of-mass energy of 13 TeV is presented, using 36.1 fb-1 of integrated luminosity collected by the ATLAS detector at the Large Hadron Collider. Events are selected in channels with two same- or opposite-sign leptons (electrons or muons), three leptons or four leptons, and each channel is further divided into multiple regions to maximize the sensitivity of the measurement. The ttZ and ttW production cross sections are simultaneously measured using a combined fit to all regions. The best-fit values of the production cross sections are σttZ=0.95±0.08stat±0.10syst pb and σttW=0.87±0.13stat±0.14syst pb in agreement with the Standard Model predictions. The measurement of the ttZ cross section is used to set constraints on effective field theory operators which modify the ttZ vertex.Peer Reviewe

Search for invisible Higgs boson decays in vector boson fusion at s=13TeV with the ATLAS detector

We report a search for Higgs bosons that are produced via vector boson fusion and subsequently decay into invisible particles. The experimental signature is an energetic jet pair with invariant mass of O(1)TeV and O(100)GeV missing transverse momentum. The analysis uses 36.1 fb of pp collision data at s=13TeV recorded by the ATLAS detector at the LHC. In the signal region the 2252 observed events are consistent with the background estimation. Assuming a 125GeV scalar particle with Standard Model cross sections, the upper limit on the branching fraction of the Higgs boson decay into invisible particles is 0.37 at 95% confidence level where 0.28 was expected. This limit is interpreted in Higgs portal models to set bounds on the WIMP–nucleon scattering cross section. We also consider invisible decays of additional scalar bosons with masses up to 3TeV for which the upper limits on the cross section times branching fraction are in the range of 0.3–1.7pb.Peer Reviewe

Search for the Higgs boson decays H → ee and H → eμ in pp collisions at s=13TeV with the ATLAS detector

Searches for the Higgs boson decays H→ee and H→eμ are performed using data corresponding to an integrated luminosity of 139fb collected with the ATLAS detector in pp collisions at s=13 TeV at the LHC. No significant signals are observed, in agreement with the Standard Model expectation. For a Higgs boson mass of 125 GeV, the observed (expected) upper limit at the 95% confidence level on the branching fraction B(H→ee) is 3.6×10 (3.5×10) and on B(H→eμ) is 6.2×10 (5.9×10). These results represent improvements by factors of about five and six on the previous best limits on B(H→ee) and B(H→eμ) respectively