Page 190 - 2019_03-Haematologica-web

N. Borràs et al.
from c.7082-2A>G in 6% and transcripts from c.8155+3G>C in 95% of reads. In platelet analyses, NGS detected transcripts without exon 6 resulting from the c.546G>A mutation in 11% of the total reads but no aber- rant transcript derived from the mutation c.7082-2A>G indicating that NMD rate could differ between 5' and 3' regions of mRNA. These results indicate the c.[546G>A; 7082-2A>G] allele is under-represented compared to the allele carrying the c.8155+3G>C mutation, showing that this allele underwent NMD in both cell types, as was pre- viously hypothesized.7
Discussion
This study reports the results of in-depth analysis of 18 PSSM in samples from VWD patients. The novel and robust procedure used, combining two-step RT-PCR and NGS sequencing, was faster and more sensitive than the method used in our previous article.7 This new approach provides several advantages, such as allele-specific indi- vidual sequences, higher sensitivity to detect transcript variants present at low copy numbers, and simplified sam- ple preparation. Moreover, the NGS data on total reads obtained for each transcript is additional information that can provide an approximation of the expression levels of each VWF mRNA. Studies are currently ongoing to con- firm that the relative expression of each transcript obtained by NGS is comparable to that provided by real- time RT-PCR.
The proven usefulness of leukocyte analysis to interpret mutations masked by NMD in platelets was seen in rela- tion to the c.3379+1G>A mutation, which had been pre- viously investigated by RT-PCR in platelets.23 In that study, the true pathogenic effect of this mutation could not be determined due to NMD. In the present study, using leukocyte mRNA and NGS technology, we found that the mutation induces two aberrant transcripts.
Similarly, the pathogenic mechanism of 7 PSSM (c.1533+1G>A, c.5664+2T>C, c.7082-2A>G, c.546G>A, c.7437G>A, and p.Cys370Tyr) was determined in leuko- cytes, and only the c.3223-7_3236dup mutation effect was observed in both cell types. Because this mutation is locat- ed in the D3 domain implicated in multimerization, it is possible that the pathogenic mechanism leading to type 1 VWD may be related to impaired VWF secretion due to intracellular retention, as has been described for other D3 mutations such as p.Cys1130Phe.24 To confirm this hypothesis, expression studies by means of heterologous cell lines or blood outgrowth endothelial cells (BOECs) will remain the gold standard.
As would be expected, mutations located in the consen- sus splicing sequence affected mRNA processing. These included c.1533+1G>A, c.5664+2T>C, leading to exon skipping, and c.7082-2A>G, activating a cryptic splice site. Both these molecular effects have been reported previous- ly in splicing mutations causing VWD.7,25,26 The c.7082- 2A>G in trans with p.Leu150Pro leads to the development of type 3 VWD. The p.Leu150Pro mutation does not affect splicing. However, other mutations located in the propep- tide, such as p.Asp141Tyr, have been identified in type 3 patients,27 and, as has been described in these cases, we suspect that p.Leu150Pro may compromise propeptide folding and affect intracellular survival and the capacity to mediate multimerization. The c.1533+1G>A mutation in
leukocytes generated three aberrant transcripts. One of them, which leads to the inframe deletion of exons 13-14, should have been detected in platelets since it would not generate a PTC and would not be degraded. However, because the mutation is located in a region with weak splicing signal sequences, as previously demonstrated,7,25,26 we suggest that the results obtained in leukocytes could be an artifact that would not occur in the natural cellular type of VWF expression. Based on these observations, we propose that the real effect of c.1533+1G>A is the skip- ping of exon 13, which predicts a frameshift at position 478 and addition of 138 aberrant amino acids before a PTC is encountered (p.Gly478AlafsTer138).
Two synonymous mutations included in our study, c.546G>A and c.7437G>A, affect splicing of VWF mRNA. To our knowledge, only three synonymous mutations affecting VWF mRNA processing have been reported: c.7056C>T,28 c.7464C>T,29 and c.3390C>T.26 Of note, the results of the present study have almost doubled the num- ber of reported mutations of this type causing VWD. The pathogenic effect of c.546G>A could only be documented by NGS; it eluded Sanger detection because of the low expression in leukocytes and platelets. The c.7437G>A mutation located at the last nucleotide of exon 43 was found in a patient with severe type 1 VWD (UMP14) in trans with c.6699_6702dup (p.Cys2235ArgfsTer8). This synonymous mutation produced 2 splicing variants. First, creation of a new DSS 4 nucleotides upstream of the WT- DSS in exon 43 that generated a PTC in exon 44 and would lead to NMD in platelets; and second, exon 43 skip- ping and generation of a PTC in exon 52. In this latter case, we would expect that NMD had been abolished in platelets, since this cellular mechanism is not effective when a PTC is encountered within 50 bp of the last exon- exon junction.30
Study of the p.Cys370Tyr (c.1109G>A in exon 9) muta- tion, resulted in generation of 2 VWF transcripts, one of them identified only by NGS because of their low expres- sion. Of note, only one other missense mutation, p.Gly1108Arg, has been reported to affect VWF splicing. Thus, these two mutations support the concept that mis- sense mutations on the last exonic nucleotides can also have repercussions on the splicing process.31 Interestingly, the pathogenic effect of this mutation is the same as that described for c.1109+2T>C (intron 9).32 In addition to the patient reported here, the p.Cys370Tyr mutation was identified in 6 additional related patients included in the PCM-EVW-ES: in homozygous state in a type 3 VWD patient and in heterozygosis in 4 type 3 carriers and 1 patient with type 1 VWD, based on their phenotype lev- els. Certain genetic modifiers of VWF levels33 and inter- individual variability in NMD efficiency between patients carrying identical mutations may lead to differences in the disease severity and clinical phenotype.34
Lastly, study of c.7081+6G>T, c.7730-4C>G, c.7730- 56C>T, c.8254-5T>G, c.3291C>T, c.4866C>T and c.[3426T>C; 3485_3486delinsTG] showed no visible effect on mRNA processing, although this does not neces- sarily mean that they have no effect on splicing. For instance, mRNA SNP analysis in patient UMP05 with c.7730-56C>T showed an absence of 1 allele in leukocytes and platelets, suggesting that the allele may have experi- enced NMD in both cell types or lack expression due to a mutation that was not detected by our sequencing proto- col. Of particular note, deep intronic mutations such as
596
haematologica | 2019; 104(3)