Abstract
Introduction A disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13 (ADAMTS13) is encoded by a 45kb gene on chromosome 9q34.2. Deficiency leads to Thrombotic Thrombocytopenic Purpura (TTP), which exists as immune TTP (iTTP), or congenital TTP (cTTP), an autosomal recessive disease occurring due to inherited biallelic pathogenic variants in ADAMTS13. Over 300 variants have been reported in cTTP, and here we have sought to create a comprehensive, accessible and interactive online database of all described ADAMTS13 variants in cTTP. This database integrates information on genetic, structural and clinical features, to increase understanding of ADAMTS13 variation and cTTP, supporting both clinicians and scientists alike.
Methods A comprehensive search strategy included a literature search of PubMed, conference abstracts from ASH and ISTH annual congresses, and databases (ClinVar, UniProt, gnomAD and the UK TTP registry), to identify ADAMTS13 variants in cTTP. cTTP was defined as ADAMTS13 activity level <10%, absence of inhibitory autoantibodies, and the presence of biallelic pathogenic ADAMTS13 variants. Information collected included DNA and amino acid changes, intron and exon location, mutation type and effect, allele frequency, geographic distribution, age at presentation and ADAMTS13 activity. Variants were mapped to X-ray and AlphaFold structures for visualisation. Predicted pathogenicity was assessed using PolyPhen-2 and FATHMM; solvent accessibility was calculated using DSSP. All data was compiled within an HTML/PHP/JavaScript-based database to allow interactive access.
Results Genetic Variation:
365 variants were identified in ADAMTS13, with 335 (92%) in exonic regions. Variants were distributed across the gene, but enriched in exon 7 (genomic position c.685-822, amino acids 230-274, within the metalloprotease domain), where 32 variants were found (11 of which were deletions). Of 190 variants with population data in gnomAD, the median allele frequency was 2.95x10-6 (range: 6.20x10-7 to 2.82x10-3), with no significant variation between variant subtype.
Protein Effects:
The most common variant type was nucleotide substitution leading to missense or nonsense amino acid changes (n=236). The most frequent amino acid changes were glutamine to a termination codon (n=13), cysteine to arginine (n=12), tryptophan to a termination codon (n=12) and arginine to cysteine (n=11). Just under half of variant amino acids (108, 46.3%) were variants from just three amino acids: cysteine, arginine and glycine. The differing R-group properties of these three amino acids highlights that a variety of amino acid changes can underlie an alteration in ADAMTS13 function.
AlphaFold and X-Ray crystal structures were included on the homepage and variant pages of the database, to allow visualisation of the variant locations. PolyPhen-2 predicted 160 (83%) missense variants as possibly/probably damaging. FATHMM analysis showed particularly damaging effects in the catalytic domain (median score of -2.31, scores < -1.5 are consistent with a damaging effect).
Clinical Features:
Age of onset of was reported for 190 variants: most patients presented in infancy or childhood (112, 58.9%) or pregnancy (72, 37.9%), with just 23 variants (12.1%) being described as an adult-onset of cTTP. Variants were reported from all continents, with European and Asian variant reports being most frequent (53% and 32% of reports respectively). ClinVar classifications were available for 78 variants, with 67% classified as pathogenic or likely pathogenic.
Conclusion This database allows visualisation of the location and effect of variants within ADAMTS13, and gives insight into DNA changes, population genetics and protein functional effects. Within the DNA changes identified, an increased frequency of variation within exon 7, encoding part of the metalloprotease domain, has not been described previously, and represents a hotspot of genetic variation in ADAMTS13. Amongst the missense variants, the high frequency of a change from cysteine to arginine, or vice versa, also highlights the deleterious effect of an alteration between a positively charged hydrophobic residue to a polar amino acid within ADAMTS13. Finally, the database has the facility for new variants to be submitted and updated over time, to allow it to expand and provide a useful, freely accessible, resource for the future.
