Page 279 - Haematologica April 2020

c.2269_2270del vWF and non-canonical splicing site
stored (in α-granules in platelets, or in Weibel-Palade bodies in endothelial cells) or secreted immediately in the circula- tion.5-7 After its secretion, vWF is recognized by the prote- olytic enzyme ADAMTS13 (a disintegrin and metallopro- teinase with a thrombospondin type 1 motif, member 13), which promptly cuts the multimeric molecule, giving rise to the oligomer pattern characteristic of vWF.8-10 Reduced lev- els of vWF, or abnormalities in vWF, are associated with von Willebrand disease (vWD), the most common inherited hemorrhagic disorder. vWD is classified as type 1 (a partial quantitative vWF defect, with a normal or almost normal multimer organization and function), type 3 (total absence of vWF), or type 2 (which includes various structural/func- tional vWF abnormalities and is further classified as types 2A, 2B, 2M or 2N, depending on which vWF domain is impaired).11 VWD is caused by mutations in the vWF gene,12 but many other genetic and environmental factors have a role in modulating circulating vWF levels, including ABO blood group, age, infections, stress, and hormone therapy.13- 15 The increasing use of genetic analysis (vWF gene sequenc- ing) for diagnostic purposes is improving our ability to char- acterize vWD, especially in cases of type 1 vWD, which is the most difficult to diagnose correctly. Numerous muta- tions have emerged as causes of vWD, along with frequent cases of multiple vWF defects, and even more complex genetic pictures in which both quantitative and qualitative vWF abnormalities co-exist.12,16 vWF cDNA sequencing showed that apparently silent mutations can activate cryp- tic splice sites or alter regulatory splicing elements, inducing exon skipping or RNA sequence deletions that often result in the formation of premature termination codons (PTC).17,18 These aberrant vWF mRNA can undergo nonsense-mediat- ed mRNA decay (NMD), a mechanism that triggers the degradation of mutated transcripts bearing PTC to avoid the synthesis of truncated proteins potentially deleterious to the cell.19,20
The above-described processes all contribute to making vWD a very complex and heterogeneous disorder, and new cases often pose a real diagnostic challenge. Here we describe a very particular vWD phenotype that is due to an equally particular genetic condition. The causative c.2269_2270del mutation was found to abolish vWF pro- duction, acting through a complex mechanism that also includes the recognition of a cryptic non-canonical splicing site in the vWF gene. To our knowledge, this is the first work to study the involvement of this rare, and still poorly understood, mechanism in the pathogenesis of vWD.
Methods
Patients
The patient investigated (proband) is a 50-year-old male with a lifelong history of severe bleeding. His 70-year-old mother and 76- year-old father, and his offspring (an 18-year-old daughter and a 15-year-old son) were also investigated. The subjects’ bleeding scores (BS) were calculated with the International Society on Thrombosis and Haemostasis (ISTH) Bleeding Assessment Tool (BAT).21 All subjects gave written informed consent in accordance with the Declaration of Helsinki.
Hemostatic analysis
Basic hemostatic tests were performed as explained elsewhere.22-24 For more details, see also the Online Supplementary Methods. The proband’s samples were diluted less than the control
plasma to adjust for his markedly reduced vWF levels. vWF multi- mers were analyzed according to previously reported proce- dures.25 Desmopressin (DDAVP) was administered subcutaneous- ly at a dose of 0.3 μg/kg 26 Anti-vWF antibody detection was per- formed by ELISA.27 ADAMTS13 activity was ascertained using FRETS-vWF73 (Peptide International, Lexington, KY, USA) as a substrate. A negative test was defined as a titer ≤16IU/mL.
Genetic analysis
The patients’ genomic DNA was obtained from whole blood; total RNA was extracted from platelets (see the Online Supplementary Methods for more details). Sequencing reactions were performed in an ABI3130 XL Genetic Analyzer.
Digital droplet polymerase chain reaction
The digital droplet polymerase chain reaction (ddPCR) assays were performed with the QX200 ddPCR system (Bio-Rad, Hercules, CA, USA) using the ddPCRTM Supermix for Probes (Bio- Rad), with different primer pairs and probes specifically designed for aberrant mRNA sequences (Online Supplementary Methods). The assay was run in multiplex, starting from 25 ng of the proband’s cDNA, at T=56°C and adding 2% DMSO to the PCR mix. cDNA was obtained by retrotranscribing RNA extracted from the proband’s platelets, as explained above.
In vitro expression experiments
The pRc/CMV-vWF vector containing the wild-type (WT)
human full-length vWF cDNA was mutated by site-directed muta- genesis using the QUIKCHANGE II XL kit (Stratagene, La Jolla, CA, USA). The primer sequences used for mutagenesis are listed in the Online Supplementary Methods. The vectors were used to transiently transfect Human Embryonic Kidney 293T (HEK293T) cells. Transfections were performed in T25 flasks using the Lipofectamine 2000 transfection reagent (Life Biotechnology, Carlsbad, CA, USA) and 4 μg of vector/T25. After 48 hours of transfection, the media containing the recombinant vWF (rvWF) were collected, and the rvWF was quantified with ELISA. The rvWF intracellular content was also measured after lysing cells with a 2% Triton-X-100 solution, as previously reported.17 The results of each transfection experiment were calculated as the mean of three replicates.
SDS-PAGE and western blot
rvWF samples were run in reducing conditions using the XCell SureLock Mini electrophoresis system (Invitrogen, CA, USA) on a Novex Tris-Glycine 3-8% precast gel (Invitrogen). After blotting, the membrane was incubated with a polyclonal rabbit anti-vWF (DAKO, Glostrup, Denmark), and then with a polyclonal anti-rab- bit-HRP antibody (for further details see the Online Supplementary Methods).
Results
Characterization of a complex von Willebrand disease phenotype
The proband’s main symptoms included epistaxis, oral cavity bleeding, frequent hematomas, and bleeding from minor wounds. He also reported episodes of hemarthrosis and severe hemorrhagic complications after tooth extrac- tions. His BS was 30 (vs. 0-3 in normal males). His mother and father (who were consanguineous) were almost asymptomatic, and so were his offspring. Hemostatic tests on the proband revealed a severely impaired hemostatic picture, with a very prolonged activated partial thrombo-
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