The effect of single nucleotide polymorphisms and mutations on congenital thrombotic thrombocytopenic purpura phenotype

Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening disease with a reported incidence of 6 cases per million per year in the UK. It is characterised by episodes of microangiopathic haemolytic anaemia and thrombocytopenia, with the widespread presence of platelet-rich thrombi in the microcirculation, leading to end-organ damage. TTP is a clinically heterogeneous disorder caused by autoantibody inhibition or clearance or by a deficiency in activity or secretion of the von Willebrand factor cleaving
protease (ADAMTS13). Over 100 mutations have been identified in ADAMTS13 yet, in some cases, these mutations in congenital TTP alone do not explain the disease phenotype, particularly in late-onset congenital TTP. One aim of this study was to analyse the effect of single nucleotide polymorphisms (SNPs), in conjunction with ADAMTS13 mutations, which have been identified in a cohort of TTP patients with varied clinical phenotype.
Twenty mutant expression plasmids containing an ADAMTS13 mutation and/or SNP have been constructed by site-directed mutagenesis (SDM). Fourteen plasmids contained a new non-synonymous mutation (C3484T) which has been rectified by further SDM. After full sequencing of the plasmid ADAMTS13 insert, 24 clones were identified as viable and the resultant protein was expressed in HEK293T cells and analysed by Western blot and ADAMTS13
antigen ELISA. Blots were scanned by densitometry and the intensity of the protein species corresponding to ADAMTS13 determined using ImageJ. The predicted effects of mutations and/or SNPs were annotated by the in silico
computational tools SIFT, PolyPhen2, I-Mutant 2.0 and SNPEffect 4.0.Results from this genotype study were compared to clinical phenotype by correlating data held within the TTP Registry at University College London.
The resultant analysis showed the mutations located in the N-terminal end of ADAMTS13 protein induced more secretory defects and SNPs had less of an effect on phenotype. The p.R7W and p.A1033T SNPs showed more of an
effect on secretion of ADAMTS13 when the mutation was located in the Cterminal end (p.R1060W) which is associated with late-onset phenotype. However, other environmental factors such as changes in the ADAMTS13-VWF axis during pregnancy and infection make a correlation between genotype and phenotype in TTP more challenging.