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N. Borràs et al.
present in ̴ 14% of reads in both cell types (Online within exon 42. This led to deletion of the 7 initial
Supplementary Table S4).
Combined potential splice site mutations in individual patients
The c.7082-2A>G mutation (intron 41) was identified in a type 3 VWD patient (UMP12) combined in trans with the p.Leu150Pro (c.449C>T, exon 5) mutation. To study c.7082-2A>G, the exon 38-43 region was amplified in platelets and leukocytes, which yielded a band of the expected size. However, Sanger and NGS sequencing of the PCR products revealed activation of a cryptic ASS
A
B
nucleotides of exon 42 (r.7082_7088del), as was predicted by the 4 algorithms, and resulted in a frameshift and a PTC (p.Ala2361GlyfsTer40) (Figure 4). Additionally, NGS showed that the aberrant transcript was present in 24% of reads. On sequencing of platelet amplicons, there were no changes, a finding highly suggestive of NMD. As type 3 VWD is characterized by absent or non-functional expres- sion of both VWF alleles, we postulated that the p.Leu150Pro missense mutation could have an effect on VWF mRNA. Nonetheless, no sequence change was iden- tified. These results were confirmed by analysis of 2
Figure 4. Analysis of the c.7082-2A>G mutation in patient UMP12. Agarose gel electrophoresis results of RT- PCR amplification of exon 38 to 43 using RNA from leuko- cytes and platelets were the same as those of healthy controls (data not shown). A) Traditional Sanger sequenc- ing of PCR product from patient leukocyte (L) demon- strate activation of a cryptic splice site 7 nucleotides downstream of the native splice site within exon 42. In patient platelet (P), only the allele carrying the p.Leu150Pro mutation could be amplified. B) NGS of PCR products from leukocytes showed deletion of the 7 ini- tial nucleotides of exon 42. Arrows show aberrant tran- scripts. C) Schematic repre- sentation of the mutation in genomic DNA and its effect on the VWF mRNA sequence. WO: without.
C
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haematologica | 2019; 104(3)