Abstract
Genetic or acquired deficiency of ADAMTS13 causes thrombotic thrombocytopenic purpura (TTP), a condition characterized by thrombocytopenia and hemolytic anemia with microvascular platelet thrombi. Several mutations spread across the ADAMTS13 gene have been identified in patients with congenital TTP. We analyzed the ADAMTS13 gene in two Turkish brothers affected by hereditary TTP. One of them is a 23 years old male, who experienced two acute TTP episodes and is currently in prophylactic treatment with plasma infusion once a month. His sister has mild thrombocytopenia and a mild hemolytic anemia but never had any acute TTP episode.Their ADAMTS13 antigen level and activity in plasma were found to be <1% and <6% respectively. Genetic analysis revealed that both patients are double heterozygote for a 29 nucleotide deletion in exon 3 (291–319del) leading to a premature stop at codon 368 in the metalloprotease domain (Peyvandi,2004) and a single base insertion in exon 29 (4143–4144insA) in the second CUB domain resulting in the loss of the 49 amino acids (Schneppenheim,2003). To evaluate the molecular mechanism of these mutations, we cloned the ADAMTS13 cDNA into pcDNA3.1 expression vector and introduced each mutation using the wild type cDNA as template (pcDNA3.1ADAMTS13insA and pcDNA3.1ADAMTS13–29del). These constructs were transiently transfected in HEK-293 cells. Expression studies demonstrated that the 4143–4144insA mutation leads to a secretion defect resulting in a reduced protease activity, measured by CBA in cell supernatant (14% of wild type activity), as reported by Pimanda (2004).The intracellular accumulation was demonstrated by WB analysis and confirmed by immunofluorescence studies showing that rADAMTS13insA was present throughout the cytoplasm while the rADAMTS13WT was mainly localized in the perinuclear area.Expression studies of 29bp deletion showed the absence of recombinant protein in cell supernatant analyzed by WB. Previous studies showed that in mammalian cells exists a regulatory mechanism (PTC-mediated decay mechanism) that triggers the destruction of PTC-bearing mRNAs and a reduction in m-RNA levels, and this decay mechanism is intron-dependent (Zhang, 1998). The 29bp deletion leads to PTC in the metalloprotease domain. To verify whether the PTC introduced by 29bp deletion could lead to degradation and decrease of mRNA level, we have constructed a minigene extending from exon 4 to exon 6 of ADAMTS13 gene including introns 4 and 5. Subsequently this fragment was subcloned in frame into pcDNA3.1ADAMTS13–29del expression vector. Reverse transcription PCR was performed on total mRNA isolated from cells transfected with ADAMTS13-WT and ADAMTS13–29del constructs.The band obtained by RT-PCR of ADAMTS13–29del mRNA was fainter than that ADAMTS13WT (roughly 10%) suggesting that the presence of PTC due to the 29bp deletion triggers probably a decay process leading to a reduction of ADAMTS13 mRNA level. In conclusion, our work shows how the association of two different ADAMTS13 gene mutations could lead to a severe ADAMTS13 deficiency, with effects on two different levels: a partial impairment of mRNA synthesis due to 29bp deletion, and an alteration of the secretion pathway caused by 4143–4144insA.
Molecular Markers of Ionizing Radiation-Induced Gene Mutations in Mammalian Cells
