ABSTRACT
INTRODUCTION: Thrombotic thrombocytopenic purpura (TTP) is a clinical thrombotic microangiopathy (TMA) syndrome defined by the pentad of symptoms. Therapeutic plasma exchange with plasma replacement is an ASFA Category I modality that can reduce morbidity and mortality if initiated early. We describe a 14-year review of patients referred for plasma exchange with a suspected diagnosis of TTP. METHODS: For 70 patients referred for urgent plasma exchange, clinical, therapeutic, and laboratory data were retrospectively analyzed, and the diagnosis was determined. RESULTS: Fifteen of the patients were diagnosed with TTP based upon ADAMTS-13 activity with the other 51 patients having other non-TTP TMA diagnoses. The mortality rate was significant for both TTP and non-TTP TMAs. PLASMIC scores were also calculated retrospectively and were noted to have limited value. TMA is a diagnostic challenge and encompasses different syndromes with similar presentations. CONCLUSION: Determining an accurate diagnosis, including prompt ADAMTS-13 testing, makes it possible to initiate appropriate therapy for the multiple different TMAs that can be seen in clinical practice.
Subject(s)
Purpura, Thrombotic Thrombocytopenic , Thrombotic Microangiopathies , Humans , Purpura, Thrombotic Thrombocytopenic/diagnosis , Purpura, Thrombotic Thrombocytopenic/therapy , Retrospective Studies , ADAMTS13 Protein , Thrombotic Microangiopathies/diagnosis , Thrombotic Microangiopathies/therapy , Plasma Exchange , SyndromeABSTRACT
The E3 ubiquitin ligase RNF168 is a DNA damage response (DDR) factor that promotes monoubiquitination of H2A/H2AX at K13/15, facilitates recruitment of other DDR factors (e.g. 53BP1) to DNA damage, and inhibits homologous recombination (HR) in cells deficient in the tumor suppressor BRCA1. We have examined the domains of RNF168 important for these DDR events, including chromosomal HR that is induced by several nucleases (I-SceI, CAS9-WT and CAS9-D10A), since the inducing nuclease affects the relative frequency of distinct repair outcomes. We found that an N-terminal fragment of RNF168 (1-220/N221*) efficiently inhibits HR induced by each of these nucleases in BRCA1 depleted cells, and promotes recruitment of 53BP1 to DNA damage and H2AX monoubiquitination at K13/15. Each of these DDR events requires a charged residue in RNF168 (R57). Notably, RNF168-N221* fails to self-accumulate into ionizing radiation induced foci (IRIF). Furthermore, expression of RNF168 WT and N221* can significantly bypass the role of another E3 ubiquitin ligase, RNF8, for inhibition of HR in BRCA1 depleted cells, and for promotion of 53BP1 IRIF. We suggest that the ability for RNF168 to promote H2A/H2AX monoubiquitination and 53BP1 IRIF, but not RNF168 self-accumulation into IRIF, is important for inhibition of HR in BRCA1 deficient cells.
Subject(s)
DNA Damage , Recombinational DNA Repair , Ubiquitin-Protein Ligases/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , BRCA1 Protein/antagonists & inhibitors , Cell Line , Chromosome Breakage , Conserved Sequence , DNA Repair , DNA-Binding Proteins/antagonists & inhibitors , Deoxyribonucleases/metabolism , Histones/metabolism , Humans , Mice , Protein Structure, Tertiary , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
BACKGROUND: RNF168 promotes chromosomal break localization of 53BP1 and BRCA1; 53BP1 loss rescues homologous recombination (HR) in BRCA1-deficient cells. RESULTS: RNF168 depletion suppresses HR defects caused by BRCA1 silencing; RNF168 influences HR similarly to 53BP1. CONCLUSION: RNF168 is important for HR defects caused by BRCA1 loss. SIGNIFICANCE: Although RNF168 promotes BRCA1 and 53BP1 localization to chromosomal breaks, RNF168 affects HR similarly to 53BP1. The RING finger nuclear factor RNF168 is required for recruitment of several DNA damage response factors to double strand breaks (DSBs), including 53BP1 and BRCA1. Because 53BP1 and BRCA1 function antagonistically during the DSB repair pathway homologous recombination (HR), the influence of RNF168 on HR has been unclear. We report that RNF168 depletion causes an elevated frequency of two distinct HR pathways (homology-directed repair and single strand annealing), suppresses defects in HR caused by BRCA1 silencing, but does not suppress HR defects caused by disruption of CtIP, RAD50, BRCA2, or RAD51. Furthermore, RNF168-depleted cells can form ionizing radiation-induced foci of the recombinase RAD51 without forming BRCA1 ionizing radiation-induced foci, indicating that this loss of BRCA1 recruitment to DSBs does not reflect a loss of function during HR. Additionally, we find that RNF168 and 53BP1 have a similar influence on HR. We suggest that RNF168 is important for HR defects caused by BRCA1 loss.
