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Effect of VWF mutations on mRNA splicing
Table 2. Effect of VWF mutations on mRNA from leukocytes and platelets.
Mutation type NT change
AA change Exon Intron
- - 13 - - 25
Leukocyte effect
Exon 13 skipping (r.1433_1533del) Exon 25 skipping (r.3223_3379del) Activation of a cryptic site at +126 nt in exon 25 (r.3349_3379del) ‡
Exon 33 skipping (r.5621_5664del)
Exon 33+34 skipping (r.5621_5842del) ‡ No visible effect
Activation of a cryptic site +7 nt
in exon 42 (r.7082_7088del)
No visible effect
No visible effect
No visible effect
Exon 6 skipping (r.533_657del) ‡
No visible effect
No visible effect
No visible effect
Exon 43 skipping (r.7288_7437) Activation of a cryptic site at +146 nt
in exon 43 (r.7434_7437del)
No visible effect
Exon 9 skipping (r.998_1109del)
Exon 8+9 skipping (r.875_1109del)
No visible effect
Platelet effect
NMD NMD NMD
NMD
NMD
No visible effect
NMD
No visible effect No visible effect No visible effect NA
No visible effect No visible effect NA
NA
NA
No visible effect
NMD
NMD
No visible effect
Protein prediction
p.Gly478AlafsTer138 p.Pro1075ValfsTer88 p.Pro1117ValfsTer88
p.Gly1874AlafsTer32 p.Phe1875_Cys1948del - p.Ala2361GlyfsTer40
-
-
- p.Thr179ProfsTer31 -
Intronic
c.1533+1G>A c.3379+1G>A*
c.5664+2T>C
c.7081+6G>T c.7082-2A>G †
c.7730-4C>G c.7730-56C>T c.8254-5T>G
Synonymous c.546G>A† c.3291C>T c.3426T>C c.4866C>T c.7437G>A
- - 51 p.Ser182 6 - p.Cys1097 25 - p.Cys1142 26 - p.Asp1622 28 - p.Ser2479 43 -
p.Leu150Pro 5 - p.Cys370Tyr 9 -
-
- - 33 - - 41
- - 41 - - 45
- - 45
DelIns c.3485_3486delinsTG p.Pro1162Leu 26 - c.3223-7_3236dup p.Pro1079_Tyr1080insLeu
-
Missense
c.449T>C c.1109G>A
p.Val2430GlyfsTer335 p.Ser2479AlafsTer23
-
p.Glu333AlafsTer87 p.Ser292ThrfsTer87 -
GlnValAspProGluPro 25 - c.6699_6702dup p.Cys2235ArgfsTer8 38 -
(r.3477_3478instttgcaggtggaccccgagcc) (r.3477_3478instttgcaggt p.Pro1079_Tyr1080insLeu ggaccccgagcc) GlnValAspProGluPro No visible effect NMD p.Cys2235ArgfsTer8
AA:amino acid; NA: not available; NMD: nonsense-mediated decay; NT:nucleotide. *Previous functional studies by Nesbitt et al.23 †Previous functional studies by Corrales et al.7 ‡Leukocyte effect observed in a really low % of reads by NGS.
the others. This may be explained by the mutation and/or other factors not explored in the current study.
The c.3291C>T mutation (exon 25), was identified in patient UMP09 (female), as well as a novel mutation in F8 (c.1346C>G, p.Ala430Gly). Of note, her brother, who was not included in this study, also had these mutations. As both siblings experienced bleeding, and the HSF predic- tion regarding c.3291C>T interpreted creation of an exon- ic splicing silencer site that could potentially alter splicing, we hypothesized that this synonymous mutation could have an effect on VWF mRNA processing. Hence, the exon 22-26 region was amplified, but no change in the VWF cDNA sequence was observed. In addition, we test- ed whether intron 25 was retained within mature VWF mRNA (Online Supplementary Methods), but no differences compared to control leukocyte cDNA were found (data not shown). Finally, as both siblings presented similar FVIII:C levels, we investigated X chromosome inactivation in patient UMP09. The results demonstrated skewed inacti- vation of the WT X chromosome in this patient (Online Supplementary Figure S5).
Missense mutations
The mutation c.1109G>A (p.Cys370Tyr, exon 9) was identified in a type 3 VWD carrier (UMP10). The exon 6-
12 region was investigated. Whereas the expected size was observed in platelets, amplification from leukocytes resulted in 2 additional bands of ̴650 and 5̴ 00 bp (Figure 3). Sanger sequencing of leukocyte mRNA confirmed that the 650-bp band corresponded to a transcript lacking exon 9. NGS of leukocyte mRNA revealed 3 different tran- scripts: the WT, a transcript skipping exon 9 (p.Glu333AlafsTer87) (13% of reads), and a transcript skipping exons 8 and 9 (p.Ser292ThrfsTer87) (7% of reads)(Table 2). Sequencing of PCR products from platelets showed no changes, suggesting that the mutated allele had experienced NMD.
Duplication mutation
The c.3223-7_3236dup mutation, which generated an in-frame tandem duplication of 21 nucleotides, including 7 nucleotides from intron 24 and the first 14 from exon 25, was detected in a type 1 VWD patient (UMP11). Study of this mutation by the two techniques demon- strated a change in the native ASS of intron 24 in both cell types, which resulted in maintaining the 21-bp insert cor- responding to 7 aberrant amino acids in mature VWF mRNA (p.Pro1079_Tyr1080insLeuGlnValAspProGluPro) (Table 2 and Online Supplementary Figure S6). Furthermore, NGS showed that the aberrant mRNA transcript was
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