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Effect of VWF mutations on mRNA splicing
c.6599-20A>T have been found to cause VWD.35 In patient UMP07 carrying the c.8254-5T>G mutation, no informa- tive SNP was identified and we were unable to determine whether there was a lack of expression of 1 allele, which could explain cosegregation of the mutation in the family with bleeding symptoms.
Study of c.[3426T>C; 3485_3486delinsTG] and c.7730- 4C>G showed no visible effect on splicing. However, these mutations have been identified in homozygous state in a type 3 patient (c.[3426T>C; 3485_3486delinsTG])3 and in a severe type 1 VWD patient (c.7730-4C>G)4 with VWF:Ag at 7% and VWF:RCo at 5%, combined with the heterozygous p.Ala631Val (previously reported in a healthy control).36 Based on these findings, there is sub- stantial evidence that these mutations would have an effect on VWF levels. Therefore, to unequivocally deter- mine the potential deleterious effect of these variants, functional studies remain essential. These studies are tra- ditionally carried out by in vitro analyses performed using heterologous cell lines (COS7, AtT-20 and HEK293). However, the advent of the possibility of obtaining BOECs from patients represents a valuable alternative,
37 since this is the functional expression site of VWF.
Moreover, BOECs allows protein expression and mRNA studies simultaneously.
In silico algorithms used to assess the impact of mutations on splicing are more sensitive and accurate in determining the putative effect of intronic mutations than that of syn-
onymous or missense mutations, such as c.546G>A and p.Cys370Tyr. Therefore, the results here should not be con- sidered definitive, and as with all analytical approaches, should form one aspect of a wider investigation.5
In conclusion, we present an extensive study reporting the effect of 18 candidate mutations on VWF mRNA pro- cessing. In vivo mRNA studies incorporating NGS technol- ogy together with traditional sequencing enabled us to determine the pathogenic effect of 8 PSSM (44%). Our study emphasizes the importance of examining selected mutations, including synonymous and missense muta- tions, to determine their pathogenic role in splicing. Taken together, our results add to the current knowledge about the molecular events leading to VWD.
Acknowledgments
We are indebted to Baxalta US Inc., now a part of Shire, for support of the PCM-EVW-ES (Grant H13-000845).
Funding
This study was also supported by the Spanish Ministry of the Economy and Competitiveness (MINECO, Ministerio de Economiá y Competitividad), Instituto de Salud Carlos III (ISCI- II) (PI12/01494, PI15/01643 and RD12/0042/0053). We are very grateful for the kind collaboration of the participating patients and their families. CIBERCV is an initiative of ISCIII, co-financed by the European Regional Development Fund (ERDF), “A way to build Europe”.
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