An 18 year-old Turkish man presented with a three month history of fatigue, nose and gum bleeding. He was found to have pancytopenia at his local Hospital and was referred to the Haematology Department of the Cerrahpasa Medical Faculty (University of Istanbul) in August 2001 for further management. His past medical history was unremarkable and there was no recent exposure to chemicals or drugs. Physical examination was unremarkable except for marked pallor and petechiae. Spleen was not palpable. His initial investigations were as follows: Hb 4.1 g/dl, WBC 1.4 × 109/l (0.6 × 109/l neutrophils), platelets 10.0 × 109/l, reticulocytes 0.4%, MCV 115.4 fl. Blood film showed anisocytosis with prominent poikilocytes and polychromasia. The serum iron, iron binding capacity, vitamin B12, folic acid and haptoglobin measurements were within the normal range. His LDH and indirect bilirubin were slightly elevated.
Bone marrow biopsy revealed variable cellularity between <5% to 60%. Cellular parts of the marrow displayed focal erythroid hyperplasia with megaloblastoid and dyserythropoietic features. Myelopoiesis was relatively reduced and some megakaryocytes were present.
Direct Coombs test was negative. Hemosiderinuria was detected. Flow cytometric analysis revealed absence of CD55 and CD59 on all granulocytes consistent with a diagnosis of PNH. He was initially treated with prednisolone (1 mg/kg/day). A moderate increase in the Hb level and reticulocyte counts (4.6%) was noted lasting a few weeks. No improvement was seen in the leucocyte and platelet counts. In November 2001 his blood count was as follows: Hb 4.7 g/dl, WBC 2.4 × 109/l, platelets 17.0 × 109/l. He was cushingoid and developed steroid induced myopathy. The prednisolone was discontinued and he was supported with erythrocyte and platelet transfusions. His blood counts were January 2002 (Hb: 4.5 g/dl, WBC 2.5 × 109/l, platelets 60 × 109/l), December 2003 (Hb:5.5 g/dl, WBC 2.9 × 109/l, Hct: 16%, platelets 13 × 109/l). In July 2002 an archived DNA sample was sent to the Department of Heamatology, Hammersmith Hospital to screen for mutations in the hTERC gene. In December 2003 DNA extracted from a buccal smear was screened for hTERC mutations.
A 654 bp fragment encompassing the hTERC gene was amplified by PCR with the primer pair 5' TCATGGCCGGAAATGGAACT3' and 5'GGGTGACGGATGCGCACGAT3' using the conditions described previously . Fragments were screened for mutation by denaturing high performance liquid chromatography (dHPLC) using the Transgenomic WAVE DNA fragment analysis system. When an abnormal pattern of elution was observed, the sample was re-amplified and subjected to direct sequence analysis by BigDye chain termination cycle sequencing. The products of the reaction were run on a 3700 DNA analyser and the sequences read using the EditView automated DNA sequence viewer (Applied Biosystems Inc). Restriction enzyme digestion of PCR products was carried out according to manufacturers instructions (New England Biolabs).
Construction of reporter plasmids and mutagenesis
Primers carrying restriction sites were used for PCR with hProm867 as the template to generate a series of 5' and 3' terminal deletions with compatible ends for cloning as Xho I/Hind III fragments into the multiple cloning region of the promoter-less luciferase vector pGL3-basic (Promega, Madison, WI). A two step cloning strategy was used for site-directed mutagenesis to prevent unexpected mutations in luciferase reporter vectors; (i) an hTR 176 bp fragment (2923 wt, spanning from -107 to +69 bp) was cloned into the Xho I/Hind III sites in pCR-Script™ plasmid vector (Stratagene, La, Jolla CA), which was used as template for PCR using a QuikChange™ site-directed mutagenesis kit (Stratagene, La, Jolla CA) following the manufacturer's instructions. (ii) All mutation fragments were reconstructed into the Xho I/Hind III sites of pGL3-basic vectors and verified by DNA sequencing. Primers carrying the mutations and a second set of primers for subcloning were designed. The PCR reaction was performed with these primers and 2923 wt or 2925 wt as template to create single-site mutation constructs. The double-site mutation construct, mSp1(2), harbouring a C-101A/C-100A double substitution, or No-Sp1, harbouring both the C-101A/C-100A double substitution and an ATGT substitution in site Sp1.1 were generated in several separate PCR reactions using mSp1.2 constructs as template. Minimal promoter mutation constructs based on 2925 wt contained the PNH C-99G mutation in site Sp1.2. The following oligonucleotides were used as PCR primers:
hTR23(+69 to +46) Hind III, 5'-cgcaagcttTACGCCCTTCTCAGTTAGGGTTAG-3';
hTR25(+10 to -12) Hind III, 5'-cgcaagctTCCGCAACCCGGTGCGCTGCCG-3';
hTR29(-107 to -88) Xho I, 5-gcgctcgAGCCCGCCCGAGAGAGTGAC-3';
hTR29Fm(-107 to -88) Xho I, 5'-gcgctcgAGCCCGCCGGAGAGAGTGAC-3';
Nucleotides corresponding to promoter sequences are given in uppercase letters from 5' end to 3' end. Lowercase letters indicate clamps for introduction of restriction enzyme sites. Bold indicates mutation.
Transfection and dual-luciferase reporter assay
3.0 μg of hTERC promoter plasmids containing firefly luciferase reporters were cotransfected into 5637 bladder cancer cells with an internal Renilla luciferase control, pRL-SV40 (Promega) using Superfect Transfection Reagent (Qiagen) as previously described [10, 11]. The total amount of DNA was kept constant at 10 μg with Salmon sperm DNA. The activity of both firefly and Renilla luciferase was determined 48 h later using the Dual Luciferase Assay kit (Promega). A minimum of three independent transfections were performed in duplicate and specific hTERC promoter activity was normalized to protein as described elsewhere [10–12].
Electrophoretic mobility shift assays (EMSAs)
Nuclear extracts from cultured 5637 cells were made according to our previous study . EMSAs were performed using the EMSA kit (Promega, E3300). 5.0 μg of nuclear extract proteins were incubated in 15 μl of reaction containing 4% glycerol, 1 mM MgCl2, 0.5 mM dithiothreitol (DTT), 0.5 mM EDTA, 50 mM NaCl, 10 mM Tris-HCl, pH7.5 and 2.0 μg poly(dI-dC) with or without 100-fold molar excess of unlabeled DNA competitors on ice for 15 min, followed by addition of the radiolabelled probe. All DNA-protein complexes were resolved by electrophoresis on 5% native polyacrylamide.