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Effect of VWF mutations on mRNA splicing
informative SNPs, rs1800375 and rs1800376, which were found in heterozygous state in leukocyte transcripts and homozygous state in platelets.
The c.[3426T>C; 3485_3486delinsTG] mutations (exon 26) were found in a type 2A VWD patient (UMP13), com- bined in trans with p.Cys2773Ser. In silico analysis predict- ed no impact on the splice site for c. 3485_3486delinsTG, but 2 of the 4 algorithms predicted that c.3426T>C would have an effect on splicing. The exon 25-28 region was investigated, but no effect was found on mRNA process- ing. Study of the p.Cys2773Ser mutation and 2 informa- tive SNPs (rs2228317 and rs4021576) confirmed that the 2 alleles were present, suggesting that exon 26 mutations do not have an effect on VWF splicing.
The mutations (c.7437G>A exon 43), and c.6699_6702dup (p.Cys2235ArgfsTer8, exon 38) were identified in trans in a severe type 1 VWD patient (UMP14) and studied only in leukocyte RNA. For c.7437G>A, we amplified exons 41-45, and 2 bands emerged: one with the expected size, and a smaller band of ̴̴350 bp (Figure 5). Sequencing analysis by both tech-
niques revealed 2 aberrant transcripts: one lacked exon 43 (p.Val2430GlyfsTer335) and the other showed activa- tion of a cryptic DSS within exon 43, leading to deletion of its 4 final nucleotides (p.Ser2479AlafsTer23) (Table 2). Study of c.6699_6702dup in leukocyte mRNA showed no splicing changes, but the duplication introduced 4 nucleotides that changed the reading frame and generat- ed a PTC (p.Cys2235ArgfsTer8).
The c.546G>A (p.=, exon 6) and c.7082-2A>G (intron 41) mutations in trans with c.8155+3G>C (intron 50) were detected in a type 3 VWD patient (UMP15). This patient had been described in our previous article, but at that time we were only able to identify the effect of c.8155+3G>C (p.Gly2706ValfsTer24) on splicing. On reanalysis of this case with NGS, we were able to characterize the effect of c.546G>A (p.Thr179ProfsTer31) and c.7082-2A>G (p.Ala2361GlyfsTer40) in cis in leukocyte mRNA. The results are consistent with those observed in patients UMP08 and UMP12, who harbored each mutation indi- vidually. In addition, in leukocytes, transcripts resulting from c.546G>A were detected in 2% of reads, transcripts
C
D
AB
Figure 5. Analysis of the c.7437G>A (p.=) mutation in patient UMP14, located in the last nucleotide of exon 43. A) RT-PCR products amplified with primers located in exons 41 and 45 in leukocyte RNA (L), separated on 1% agarose gel. B) Traditional Sanger sequencing of PCR product from patient leukocyte (L) shows two aber- rant transcripts: activation of a cryptic splice site - 4 nucleotides upstream to WT-DSS - in exon 43 (1), and exon 43 skipping (2). C) NGS of PCR products gave the same results than Sanger sequencing. D) Schematic representation of the mutation in genomic DNA and its effect on the VWF mRNA sequence. M indicates a 100- bp DNA ladder. WO: without.
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