Page 281 - Haematologica April 2020

c.2269_2270del vWF and non-canonical splicing site
from position c.2281, at the beginning of exon 18, the chromatogram became triple, a condition not found in the patient’s genomic DNA, suggesting the co-existence of more than one mRNA species. To separate and character- ize these sequences, the PCR-amplified cDNA fragment spanning exons 15-18 was cloned into a TA-vector, then sequenced again. Three vWF mRNA species were thus identified: (i) the r.2269_2270del (RNAI), lacking the CT dinucleotide at position 2269-2270; (ii) the r.[2269_2270del;2282_2288del] (RNAII), in which the first seven nucleotides of exon 18 were also deleted (position c.2282-2288); and (iii) the r.[2269_2270del;2281_2282insAG] (RNAIII), where an AG insertion was found 11 nucleotides downstream from the CT deletion (Figure 3). The fact that neither the c.2282_2288del nor the c.2281_2282insAG mutations had been found in the patient’s genomic DNA indicates that they were the result of an altered splicing of exon 18. The deletion of 7 nucleotides in RNAII could be explained by the use of the AG nucleotides at position c.2287-2288 as the acceptor site, instead of the WT one. This hypothesis was confirmed by an in silico analysis with the Human Splicing Finder 3.1 software, which predicted the c.2287- 2288 AG dinucleotide as a potential cryptic acceptor site, with a consensus of 72.4 (in a range from 1 to 100, where 100 is the score corresponding to the normal splicing sites). On the other hand, the use of a non-canonical acceptor splicing site (the CG nucleotide pair at position c.2282-4_2282-3) might explain the retention of the c.2281-2282 AG dinucleotide in RNAIII, a pathogenic mechanism hitherto never described in vWD. The AG retention was confirmed by repeating the total RNA extraction and retrotranscription process twice, using dif- ferent enzymes.
Genotype-phenotype correlation - Three different vWF mRNA were thus found associated with the c.2269_2270del genomic mutation. The RNAI form result- ed in a truncated vWF (p.Leu757Valfs*22, indicated as P1 from now on), due to the generation of a premature trans- lation stop codon as a consequence of the c.2269-2270 CT deletion. In RNAII, deriving from the usage of a cryptic
AG splicing site, the further 7 nucleotide deletion (c.2282- 2288) restored the altered reading frame, producing a mature vWF slightly shorter than normal, and lacking the Arg763-Ser764 furin cleavage site (p.L757_R763delinsVSSQ, named P2 from now on). In RNAIII, the c.2281-2282 AG retention resulting from the use of a non-canonical CG acceptor splicing site restored the reading frame lost due to the upstream CT dinu- cleotide deletion, producing an in-frame, full-length vWF, but with an altered 757-761 amino acid sequence (p.L757_761delinsVSSQG, named P3), corresponding to the consensus binding site of furin.30 Assuming no contri- bution of RNAI to vWF production (the P1 vWF cannot dimerize), the patient’s phenotype should be due to the co-existence of two mutated vWF species (P2 and P3 vWF), both resistant to the enzymatic action of furin. In our patient, the residual synthesis of mutated vWF, char- acterized by the persistence of its propeptide, confirms the presence of these P2 and P3 vWF forms.
Different expression of RNAI, RNAII and RNAIII - A multi- plex ddPCR assay was performed on the proband’s cDNA, to examine the levels of expression of the three mRNA. RNAI was found to be the dominant species (6.3 copies/ng, 83% of the total vWF mRNA), followed by RNAII (0.96 copies/ng, 12% of the total vWF mRNA), and RNAIII (0.37copies/ng, 5% of the total vWF mRNA). That the RNAI species encoding for the truncated p.Leu757Valfs*22 vWF accounts for more than 80% of the patient’s vWF mRNA is consistent with his severe quanti- tative vWF defect.
In vitro expression experiments - To establish the effect of the single and combined aberrant mRNA on vWF synthe- sis and release, we ran in vitro experiments using HEK293T cells and vectors pRc/CMV-vWF, pRc/CMV-vWF-RNAI, pRc/CMV-vWF-RNAII and pRc/CMV-vWF-RNAIII, encoding for WT, P1, P2 and P3 vWF, respectively.
RNAI - No rvWF was found in the conditioned medium of HEK293T cells transfected with the pRc/CMV-vWF- RNAI, while co-transfection of both the WT and the mutated vector (mimicking a heterozygous condition) induced a drop in rvWF production of about 30% (Figure
Figure 2. The proband’s vWF multimer pattern before (0’) and at various times after desmo- pressin (DDAVP) administration. The proband’s multimer pattern was obtained from plasma diluted 1:2.5, instead of the 1:20 dilu- tion used for the normal plasma (NP). Throughout the test, there was no sign of any discontinuous oligomers or additional discrete bands other than the protomer and the ultralarge band, while the two bands already seen before administering DDAVP appeared significantly increased. On the other hand, there was no evidence of any increase in the aspecific band below the dimer.
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