Homozygous or compound heterozygous mutations in the ADAMTS13 gene cause hereditary thrombotic thrombocytopenic purpura (TTP), an inherited disorder characterized by severe deficiency of plasma ADAMTS13 activity resulting in occlusive microvascular thrombosis. More than eighty ADAMTS13 mutations have been identified, of which 15 have been found in homozygous state. Homozygous mutations are of special interest to unravel a correlation between the ADAMTS13 genotype and the often variable clinical phenotype in hereditary TTP. We studied 5 ADAMTS13 mutations found in homozygous state in 6 patients with hereditary TTP. Detailed patients’ histories were recorded and plasma ADAMTS13 activity and antigen levels were determined. Recombinant ADAMTS13 mutants were transiently expressed in COS-7 cells and stably transfected HEK293 cell lines were generated for all five mutants. The biosynthesis, secretion and specific enzymatic activity under both static and flow conditions were studied. Intracellular localization of the mutants was also determined by immunofluorescent microscopy. Patients homozygous for W688X, R692C or C804R became manifest with acute TTP as neonates and several siblings had died of a TTP-like disorder. Plasma ADAMTS13 activity was < 1% of the normal and ADAMTS13 antigen was undetectable. In patients with homozygous R1060W mutation, milder courses with adult onset of TTP after strong triggering factors were observed. One patient presented with the first TTP attack at the age of 19 years during her first pregnancy while a second patient experienced a first bout at the age of 61 years during a severe pneumonia. Both patients had 3% of residual plasma ADAMTS13 activity and 6% of antigen of normal human plasma. Homozygous mutation G1239V was found in a patient with frequent TTP bouts in childhood starting at 3 years of age and an attenuated course in adulthood with attacks triggered by excessive alcohol consumption. His plasma ADAMTS13 activity was <1% along with undetectable antigen. Recombinant mutants R692C, C804R, R1060W and G1239V migrated at 190 kDa like wild-type ADAMTS13. Secretion of all 4 missense mutants was severely reduced and was highest for R1060W. The truncated mutant W688X was efficiently secreted with a molecular weight of 97 kDa. This mutant could cleave the VWF73 peptide as did wild-type ADAMTS13. Proteolytic activity towards multimeric von Willebrand factor (VWF) under static conditions was moderately impaired and even substantially reduced under fluid shear stress as compared to wild-type. Immunofluorescent staining showed the mutant ADAMTS13 proteases within endoplasmatic reticulum and Golgi apparatus, consistent with the distribution of secretory proteins. We conclude that all 5 investigated ADAMTS13 mutations are causative for hereditary TTP. The homozygous missense mutations in the carboxyl-terminal part of ADAMTS13 severely impair the secretion of the protease while the truncating mutation affects the proteolytic activity towards multimeric VWF under shear stress. Our findings highlight the significance of the middle and distal carboxylterminal domains of ADAMTS13 for secretion and enzymatic activity in vivo. This study of homozygous patients showed an early onset and severe course of TTP for mutations in the 2nd and 3rd TSP-1 repeat while the mutations in the 7th TSP-1 and the first CUB domain led to a later onset and a milder course of TTP with bouts requiring strong triggering factors. The two patients homozygous for R1060W showed low levels of residual plasma ADAMTS13 activity and antigen and the mildest clinical courses which is consistent with the cell culture studies. Together, our findings suggest a phenotype-genotype correlation in hereditary TTP.

Disclosures: Lammle:Baxter: Consultancy. Kremer Hovinga:Baxter: Consultancy.

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