The first deletion mutation in the TSP1-6 repeat domain of ADAMTS13 in a family with inherited thrombotic thrombocytopenic purpura

Abstract

analysis identified a homozygous deletion of nucleotides 2930-2935 (GTGCCC) in exon 23 of ADAMTS13 in the 2 probands, but also in one asymptomatic sibling. 13 In order to explain the patients' phenotype, we then studied the mechanistic effect of the deletion by means of expression studies in mammalian cells. Design and Methods PatientsTwo South Iranian patients (2 brothers), off-spring of first cousins, were affected by chronic recurrent TTP that first developed during adulthood (Figure 1). Patient II:2, a 26 year old male, had his first episode of TTP at the age of 23 years and 6 subsequent recurrent episodes with no precipitating event or triggering agent. Bleeding symptoms such as purpura and petechiae were present at each episode, accompanied by fever and vomiting, whereas mild neurological symptoms (visual disorders and drowsiness) were observed only during the first episode. The clinical diagnosis was established at the time of the first episode by the presence of thrombocytopenia (platelet count no higher tha 20×10 9 /L), Coombs negative hemolytic anemia (Hb 10.3 g/L), fragmented erythrocytes and high serum level of lactate dehydrogenase (LDH 1055 UI/L). The patient was successfully treated during each acute episode with plasma exchange and high dose of corticosteroids, before the molecular diagnosis. After his sixth episode of TTP, to prevent further relapses, the patient started a prophylactic treatment with fresh frozen plasma (FFP) (30 ml/kg) every three weeks. Patient II:3, a 31 year old male, developed his first episode of TTP at the age of 29 years, in association with an episode of pneumonia. He had purpura and petechiae on his legs, a platelet count of 29×10 9 /L and Coombs negative hemolytic anemia (Hb 8.5 g/L, LDH 954 UI/L). Daily FFP infusions (30 mL/kg) were effective as reflected by a progressive increase in the platelet count. Since the first disease episode, the patient receives an FFP infusion when his platelet count falls below 100×10 9 /L. According to the family history, another male sibling (II:1) had a TTP episode at the age of 23 years and died because of multiorgan failure. Another 4 brothers and one sister are all healthy and have never had any signs or symptoms of TTP. This study was carried out with the approval of the local ethics committee Ex-vivo analysisADAMTS13 activity and antigen were measured in plasma samples and in the conditioned media of cells transfected by wild type (ADAMTS13WT) and mutant (ADAMTS13del6bp) expression vectors using respectively a collagen binding assay (CBA) and an immunoassay, both modified as previously described.14 The presence of neutralizing (inhibitors) and non-neutralizing anti-ADAMTS13 autoantibodies in patients' plasma was also evaluated.14 The coding region and intron-exon boundaries of the ADAMTS13 gene (NT_017539) were amplified and sequenced as previously reported. 5 In vitro expression studiesThe complete ADAMTS13 wild-type cDNA was inserted into the mammalian expression vector pcDNA TM 3.1/V5-His TOPO ® TA (Invitrogen, Carlsbad, CA, U...

Inherited thrombotic thrombocytopenic purpura

“…In agreement with this finding, the report of Palla et al 15 shows that the recombinant 2930-2935del GTGCCC mutant protein has normal specific activity. On the other hand this mutation, by substituting the C977 with a W, probably alters the folding of the protease by disrupting a disulphide bond within the sixth TSP1 domain.…”

“…Only 15 mutations have been observed in homozygous form, including the one described in this issue of the journal by Palla et al, 15 who found a homozygous deletion of nucleotides 2930-2935 in exon 23 of ADAMTS13 in a South Iranian family. Interestingly, the deletion does not result in a frameshift and in protein interruption but it causes the replacement of C977 by a W and the deletion of A978 and R979 in the sixth TSP1 repeat domain where no mutations had so far been described.…”

ADAMTS13 Secretion and Residual Activity among Patients with Congenital Thrombotic Thrombocytopenic Purpura with and without Renal Impairment

“…Of 18 patients, 10 had previously been screened for ADAMTS13 mutations by our groups (Table 1) (16,17,19,20,25,26). Here we identified the genetic ADAMTS13 defect in the other 8 patients (R002, F1518#728, F1519#728, F1627#873, F1628#873, F769#239, F1116#239, and R564).…”

“…The v aria nts p.V88 M, p .Q9 7f sX31 , p.C9 77 W +A978_R979del, p.R1123C, p.R1219W, p.G1239V, and p.E1382RfsX6 were previously described and functionally characterized by our groups (Table 3) (19,20,25). The p.R1060W has already been expressed by Camilleri (27) and Tao (28) and colleagues; however, these investigators reported different levels of secretion (,5% and 11.4%, respectively) and activity (fully active and 35% active, respectively).…”

Amino acid residues Arg659, Arg660, and Tyr661 in the spacer domain of ADAMTS13 are critical for cleavage of von Willebrand factor

“…42 A deletion mutation in the TSP1 sixth repeat of ADAMTS13 was identified in a family with hereditary TTP. 43 Furthermore, a homozygous mutation (W688X) found in a patient with hereditary TTP, which truncates ADAMTS13 4 amino acids after the spacer domain, was reported to have comparable activity to catalyze VWF73 peptides and predenatured VWF with wild-type ADAMTS13 but exhibited much less activity in cleaving native VWF under fluid shear stress. 44 Finally, autoantibodies against the TSP1 second to eighth repeats and the CUB domains of ADAMTS13 were identified in many patients with acquired TTP, although the functional consequence of these anti-ADAMTS13 IgGs remains to be determined.…”