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Effect of VWF mutations on mRNA splicing
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Figure 2. Analysis of the c.546G>A mutation, located at nucleotide 14 from the beginning of exon 6, in patient UMP08. A) RT-PCR products amplified with primers located in exons 4 and 7 in leukocyte RNA (L), separated on 1% agarose gel. B) Traditional Sanger sequencing of PCR product from patient L (1) showed the single nucleotide change. C) NGS of PCR products from leukocytes identified the single nucleotide variant, as well as exon 6 skipping. Arrows show aberrant transcripts. D) Schematic representation of the mutation in genomic DNA and its effect on the VWF mRNA sequence. M indicates a 100-bp DNA ladder.
transcripts: the major one (37% of reads) showed exon 33 skipping, and the minor (2% of reads) showed exons 33 and 34 skipping. Moreover, analysis of the p.Leu2407Pro mutation confirmed that NMD of the allele carrying the c.5664+2T>C mutation had occurred in platelets.
Candidate intronic mutations
The c.7081+6G>T mutation, identified in a type 1 VWD patient (UMP04), generated a new GT dinucleotide in intron 41 (Online Supplementary Table S3). The exon 38-43 region showed the expected PCR product in both cell types (772 bp). To confirm these results, two informative SNPs (rs216321 in exon 20 and rs216902 in exon 35) were geno- typed. Both were in heterozygous state, indicating that the allele carrying the mutation was unaffected by NMD.
The c.7730-56C>T mutation (intron 45) was identified in a type 1 VWD patient (UMP05). The exon 43-49 region, examined by Sanger and NGS, showed no visible effect on mRNA splicing. To confirm the presence of the allele car- rying the intronic mutation, two informative SNPs were
analyzed: rs216321 and rs1800380. Surprisingly, the sequence obtained in both cell types indicated that only one allele was expressed. To further explore these results, we tested whether intron 45 was retained within the mature mRNA (Online Supplementary Methods) and per- formed complete sequencing of VWF cDNA from leuko- cytes. Nonetheless, no changes were observed (data not shown).
The c.7730-4C>G mutation (intron 45) was identified in a type 2A/2M patient (UMP06), combined with the p.Arg1374Cys mutation. The HSF predicted activation of a cryptic intronic acceptor splice site (ASS), but in the exon 43-49 region, splicing was not affected by the mutation. To confirm the presence of the allele carrying c.7730- 4C>G, the p.Arg1374Cys mutation was analyzed, and both nucleotides were seen in platelets and leukocytes, suggesting expression of both alleles. We then performed the same experiment as was done in patient UMP05 to test intron 45 retention, but no changes in VWF cDNA were observed (data not shown).
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