Screening for ADAMTS13 Gene Mutations in Thrombotic Thrombocytopenic Purpura Reveals a New Deletional Mutation.
Authors: Fidalgo Teresa , Martinho Patricia , Salvado Ramon , Pinto Catarina Silva , Marques Dalila , Gonçalves Elsa , Martins Natalia , Ribeiro Maria Leticia 
Published: April 2020

Abstract
Thrombotic Thrombocytopenic Purpura (TTP) is a rare severe thrombotic microangiopathy due to the presence of ultra large von Willebrand factor (UL-vWF) multimers in circulation. The aetiology has been linked to a congenital deficiency or inhibition of the vWF-cleaving protease (ADAMTS-13) activity by autoantibodies, which neutralise the enzymatic activity or bind the protease, increasing the plasmatic clearance (non-neutralising). The presence of autoantibodies can be associated with mutations and polymorphisms (SNPs) in the ADAMTS-13 gene. The goal of this study was to characterize the ADAMTS-13 in 4 female patients with acute TTP episodes. The clinical picture was characterized by neurological symptoms and microangiopathic hemolytic anemia (Hb 6 – 9 g/dL), thrombocytopenia (platelets 4 – 47 x109/L), elevated LDH (1874 – 4922 IU/mL). In one patient TTP episodes occurred during 4 pregnancies resulting in fetal deaths; one patient has systemic lupus. No precipitant factors were identified in the other 2 women; 1 of them had 3 TTP episodes.
Methodology: ADAMTS-13 antigen and IgG anti-ADAMTS-13 were studied by ELISA (American Diagnostica). The vWF multimeric pattern was analyzed by SDS electroforesis, Western Blot, imunofixation and densitometry. Mutations screening along the 29 exons and boundaries of the ADAMTS-13 gene was performed by direct sequencing.
Results: All the patients presented UL-vWF and autoantibodies anti-ADAMTS-13 and decreased ADAMTS-13 antigen. ADAMTS-13 gene studies identified SNPs in the four patients and mutations 764_776 del12 and R1096H, in heterozygosity, in the two patients with TTP associated with pregnancy and lupus, respectively.
ADAMTS13 ADAMTS13 gene Patients Age TTP UL vWF Ag (ng/ml) IgG (AU/ml) Mutation Modulators SNPs Reference range: Ag: 485–1242 ng/ul; IgG: cutoff &gt;13.9 AU/ml; *3 episodes; **4 episodes I 73 idiopatic + &lt;62.5 18 (pos) No mut R7W (Htz); Q488E (Hm); II 41 Idiopatic* + 127 20.3 (pos) No mut R7W (Hm); Q488E (Hm)&#x2028; P618A (Hm); A732V (Hm) III 32 lupus + 284 30 (pos) E25, R1096H (Htz) R7W (Htz); Q488E (Hm) IV 26 Pregnancy** + &lt;62.5 22.6 (pos) E7, 764_776del12 (Htz) R625H (Hm)
Discussion: PTT in these four patients was associated with antibodies anti-ADAMTS-13 and low levels of protein. Identification of the R7W and Q488E SNPs, described as positive modulators of protein secretion, on Patient I suggests the presence of the non-neutralising type of antibodies responsible for an increased plasmatic protein clearance. The positive modulation of the same SNPs on Patient II and III may be abolished by the presence of SNPs P618A and A732V (Camilleri et al; Pleimaueur et al) on Patient II and the mutation R1096H (Meyer et al) on Patient III. The not previously described mutation 764_776del12, identified on Patient IV, is likely to disturb the protein aminoacid composition, compromising the protease structure and function. This patient had a PTT episode in each of her four pregnancies.