PMC:6080766 / 4387-10756 JSONTXT

{"project":"2_test","denotations":[{"id":"30057030-9496720-2055525","span":{"begin":1184,"end":1186},"obj":"9496720"},{"id":"30057030-10190923-2055526","span":{"begin":1268,"end":1270},"obj":"10190923"},{"id":"30057030-24123394-2055527","span":{"begin":1584,"end":1586},"obj":"24123394"},{"id":"30057030-26299366-2055528","span":{"begin":1588,"end":1590},"obj":"26299366"},{"id":"30057030-10352172-2055529","span":{"begin":1849,"end":1851},"obj":"10352172"},{"id":"30057030-18923082-2055530","span":{"begin":2222,"end":2225},"obj":"18923082"},{"id":"30057030-10352172-2055531","span":{"begin":2239,"end":2241},"obj":"10352172"},{"id":"30057030-9388193-2055532","span":{"begin":3150,"end":3152},"obj":"9388193"},{"id":"30057030-8157639-2055533","span":{"begin":3154,"end":3156},"obj":"8157639"},{"id":"30057030-14685245-2055534","span":{"begin":3272,"end":3274},"obj":"14685245"},{"id":"30057030-29043644-2055535","span":{"begin":5139,"end":5141},"obj":"29043644"},{"id":"30057030-27984745-2055536","span":{"begin":5143,"end":5145},"obj":"27984745"},{"id":"30057030-26643143-2055537","span":{"begin":5957,"end":5959},"obj":"26643143"}],"text":"Material and Methods\n\nResearch Subjects\nGenomic DNA from the affected individuals and family members was extracted from peripheral blood by standard methods or obtained from saliva samples with Oragene collection kits according to the manufacturer’s instructions. Informed consent was obtained from all participating families, and all procedures performed in studies involving human participants were in accordance with the Declaration of Helsinki. Research studies were approved by the Scottish Multicenter Research Ethics Committee (05/MRE00/74), the Hospital Universitario La Paz Ethical Committee (PI-2630), the Great Ormond Street Hospital Research Ethics Committee (09/H0706/66), the University Medical Center Göttingen Ethics Committee (vote ref. 3/2/1), and the Institute for Genomic Medicine at Columbia University (protocol AAAO8410).\nParents provided written consent for the publication of photographs of the affected individuals. For growth measurements, Z scores (standard deviations from population mean for age and sex) were calculated according to LMS (L, smooth curve; M, median; S, coefficient of variation) growth on the basis of British 1990 growth reference data.20 For calculation of Z scores for growth measurements published for Bloom syndrome,21 full-term gestation was assumed, and postnatal growth measurements were calculated from data provided for the 18- to 21-year age range.\n\nExome Sequencing and Sanger Sequencing\nExome sequencing and confirmatory capillary Sanger sequencing were performed according to standard methodologies as previously published.22, 23 TOP3A and RMI1 variants were annotated with GenBank: NM_004618.4 and NM_024945.2 reference sequences, respectively.\n\nPlasmid Construct and Protein Purification\n\nCloning of the Mutant hTopIIIα Expression Vector\nA plasmid encoding the wild-type (WT) TOP3A cDNA24 was modified by the Quickchange XL Site-Directed Mutagenesis Kit (Agilent technologies) with the following primers to recapitulate the deletion and frameshift present in subject P1: 5′-CCCTCCGTCACACGACTGTGCAGAAGGA-3′ (T3_FS_FW) and 5′-TCCTTCTGCACAGTCGTGTGACGGAGGG-3′ (T3_FS_RW).\n\nExpression and Purification of TRR\nThe previously described plasmids encoding RMI1 and RMI213 and hTopoIIIα24 were co-transformed into E. coli Rosetta 2 cells, and the complex was expressed. The cells were disrupted in buffer A (50 mM Tris-HCl [pH 7.5], 0.5 M NaCl, 10% glycerol, 0.1% IGEPAL, 2 mM β-mercaptoethanol, 40 mM imidazole, 1 mM PMSF, and protease inhibitor tablet [PI, EDTA-free, Roche]) on ice before dounce homogenization and sonication. After the removal of cell debris by centrifugation, the lysate was affinity purified on a 5 mL HisTrap HP affinity column. The complex was further purified on a 5 mL HiTrap Heparin HP column in buffer B (50 mM Tris-HCl [pH 7.5], 10% glycerol, 0.1 mM EDTA, and 1 mM DTT) with a linear gradient of 200 mM to 1 M NaCl and then gel filtered on a 120 mL HiLoad 16/600 Superdex 200 column in buffer B containing 200 mM NaCl. Expression and purification of the mutant TThr812LeufsTer101RR complex were performed in a similar manner. BLM was purified as described previously.25, 26\n\ndHJ Dissolution\nThe dHJ substrate construction and dissolution reactions were carried out as described previously.14\n\nCell Culture\nDermal fibroblasts were obtained by skin-punch biopsy and were maintained in amnioMAX C-100 complete medium (Life Technologies) in a 37°C incubator with 5% CO2 and 3% O2. siRNA oligonucleotides (siLUC: 5′-CUUACGCUGAGUACUUCGA-3′ [siTopIIIα SMARTpool M-005279-01-0005, Dharmacon]) were transfected into dermal fibroblasts with RNAiMAX (Life Technologies) according to the manufacturer’s instructions.\n\nSister Chromatid Exchange Assay\nDermal fibroblasts were treated with 10 μM BrdU for 48 hr followed by 0.5 μg/mL colcemid for 2 hr. Metaphases and nuclei were isolated in hypotonic buffer (0.25% KCl and 1% Na3C6H5O7), fixed with methanol and acetic acid (3:1 vol/vol), and dropped onto slides. Dried slides were rehydrated in PBS and then incubated with 2 μg/mL Hoescht 33342 stain in 2× saline sodium citrate (SSC) buffer (300 mM NaCl and 30 mM sodium citrate) for 15 min. Slides were then covered in 2× SSC buffer, irradiated in ultraviolet A at 5,400 J/m2, dehydrated in an ethanol series, and mounted in VECTASHIELD with DAPI.\n\nSCE Methodology for Analysis of P7 and P8\nDermal fibroblasts were treated with 26.05 μM BrdU for 72 hr followed by 0.5 μg/mL colcemid for 5 hr and then harvested. Cells were resuspended in a pre-warmed hypotonic solution (0.051 M KCl) for 20 min at 37°C, fixed with methanol and acetic acid (3:1 vol/vol), and dropped onto slides. Samples were aged for at least 3 days and then stained with 1.56 μg/mL Hoechst 33258 (Sigma, B2883). Slides were irradiated in 2× SSC with ultraviolet C (356 nm) at 0.260 J/cm2, washed with 2× SSC, and stained with 10% Giemsa in phosphate buffer.\nSCE analysis of peripheral-blood samples was performed according to standard methodology in a diagnostic lab setting.27\n\nImmunofluorescence and microscopy\nMitotic abnormalities and G1-associated defects were analyzed as described previously.28, 29 In brief, for mitotic analysis, cells were seeded onto 22 × 22 mm glass coverslips in 6-well plates. After 18 hr, cells were treated with 3.5 μM RO3306 for 6 hr for the induction of a late G2 arrest and were subsequently released into fresh media. After 45 min, cells were fixed with 4% paraformaldehyde containing 0.2% Triton X-100 in PBS for 20 min. For G1-associated defects, RO3306-treated cells were released into fresh media for 30 min. Prometaphase cells were shaken off and re-seeded onto glass slides coated with poly-l-lysine. After 4–6 hr, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 for 10 min. Fixed cells were incubated with antibodies specific to cyclin A (Santa Cruz Biotechnology, sc-596), 53BP1 (Santa Cruz Biotechnology, sc-515841), and PICH.30\n\nImmunoblotting\nCells were lysed in 50 mM Tris-HCl (pH 8), 280 mM NaCl, 0.5% NP40, 0.2 mM EDTA, 0.2 mM EGTA, and 10% glycerol supplemented with a protease inhibitor tablet (Roche Life Science). Protein samples were run on a 4%–12% NuPAGE Bis-Tris precast gel (Life Technologies) and then immunoblotted with anti-TopIIIα raised against amino acids 652–1,001 (Proteintech, 14525-1-AP) and actin (Sigma, A2066).\n"}