Abstract
Background
Although autosomal recessive hematologic disorders are individually rare and difficult to ascertain, studies involving one or more homozygous affected children and their unaffected heterozygous parents have led to expanded understanding of known and discovery of previously unknown processes. The son and daughter of two Salvadoran parents were diagnosed with congenital thrombotic thrombocytopenic purpura (cTTP) at 6 and 2 years of age, respectively, after presenting with fever, respiratory symptoms, hemolytic anemia, and thrombocytopenia and being found to have ADAMTS13 activities <1% without neutralizing IgG antibodies. They remain without long-term neurologic or renal sequelae following prophylactic infusions of fresh plasma (10 mL/kg every 2.5-weeks). The purpose of this study was to characterize and correlate single-nucleotide variations (SNVs) in each parent's, non-mutant ADAMTS13 allele with its mRNA and protein expression, activity, and enzyme kinetics.
Methods
Prior to a plasma infusion, blood samples were collected from the children and parents. Genomic DNA was isolated for polymerase chain reaction (PCR), and direct Sanger sequencing of all ADAMTS13 exons and flanking intronic segments was performed; all variants identified were confirmed by bidirectional sequencing of a second, independently generated amplicon. Total RNA was isolated and the steady-state level of ADAMTS13 mRNA was measured using a quantitative real-time PCR (q-RT-PCR)-based assay. ADAMTS13 was characterized enzymatically using the fluorogenic FRETS-VWF73 substrate and antigenically by ELISA.
Results
Both children were found to be homozygous and parents to be heterozygous for the previously described, cTTP-causing ADAMTS13 single-base-substitution mutation 20506C>T, a missense mutation that encodes cDNA-nucleotide 2518 (c.2518C>T) and ADAMTS13 residue 692 (692Arg>Cys [692R>C]) (Fig. A). As expected, the children's ADAMTS13 antigen and activity levels were undetectable, although notably, steady-state levels of the ADAMTS13 mRNA were >2.5-fold higher in the daughter than in the son. The re-sequenced regions of the ADAMTS13 loci segregating within this family contained 26 additional SNVs, seven of which were nonsynonymous (ns) including two previously unreported ns-SNVs: 27852C>T (c.3362C>T; 972Arg>Trp) and 33325G>A (c.3733G>A; 1096Arg>His) (Fig. A, left panel). The parents' genotypes differed at nine positions, including three ns-SNVs, creating two distinct, non-mutant haplotypes (designated I and III) at the gene, mRNA and protein levels. The q-RT-PCR assay revealed >4-fold higher steady-state mRNA levels in the father compared to the mother (p<0.001; Fig. B). Plasma ADAMTS13 activity and antigen levels were ∼2-fold greater in the father than in the mother (p=0.00164 and p=0.0633, respectively), but the specific activities of these structurally distinct ADAMTS13 proteins were notably almost identical (253.5 vs. 256.2 U/μg). Moreover, initial velocity kinetic analysis using the Michelis-Menten equation demonstrated that the Vmax of the father's ADAMTS13 was twice that of the mother's (1.4 vs. 0.7; p < 0.0001) while its affinity for substrate was one-third that of her ADAMTS13 (Km = 0.3 vs. 0.1; p = 0.0585).
Discussion
We capitalized on the fortuitous finding of children with complete homozygosity across ancestrally-related ADAMTS13 alleles harboring a null-type, loss-of-function mutation, as this enabled the substantially different levels of gene expression and function observed in the parents to be attributed to their two previously unreported, SNV-based, ADAMTS13 haplotypes. Additional investigation at the molecular, biochemical, cellular, and organismal levels will be necessary to determine which of the myriad potential individual SNV- and/or haplotype-based mechanisms are responsible for the observed parental differences in circulating ADAMTS13 antigen and activity.
Disclosures:
Kim: Haplomics, Inc.: Membership on an entity’s Board of Directors or advisory committees; Baxter: Honoraria. Marder:Baxter: Research Funding. Howard:Haplomics, Inc.: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Baxter: Research Funding.
Single-nucleotide variations defining previously unreportedADAMTS13haplotypes are associated with differential expression and activity of the VWF-cleaving protease in a Salvadoran congenital thrombotic thrombocytopenic purpura family
