Page 186 - 2019_03-Haematologica-web

N. Borràs et al.
AB
C
D
Figure 3. Analysis of the c.1109G>A (p.Cys370Tyr) mutation in patient UMP10, located in the last nucleotide of exon 9. A) RT-PCR products amplified with primers located in exons 6 and 12 in leukocyte (L) and platelet (P) RNA, separated on 1% agarose gel. B) Traditional Sanger sequencing of PCR product from patient L (2) showed exon 9 skipping. The L (3) band could not be purified in the agarose gel due to low concentration, thus it was not analyzed by Sanger. In platelets, only the non-mutated allele could be amplified. C) NGS of PCR products from leukocytes identified transcripts lacking exon 9, as well as transcripts lacking exons 8 and 9. D) Schematic representation of the mutation in genomic DNA and its effect on the VWF mRNA sequence. M indicates a 100-bp DNA ladder.
The c.8254-5T>G mutation (intron 51) was found in a type 1 VWD patient (UMP07). The exon 49-52 region was amplified, and the expected 546-bp size was observed in both cell types. Sequence analysis by the two techniques revealed no changes in VWF cDNA. Interestingly, this patient’s two children, heterozygous for the c.8254-5T>G mutation, had bleeding symptoms. Hence, we performed complete sequencing of VWF cDNA from leukocytes. However, no changes were identified.
Synonymous mutations
The mutations c.546G>A (exon 6) and c.4866C>T (exon 28) were identified in trans in a type 1 VWD patient (UMP08). These mutations were studied only in leukocyte RNA. To investigate c.546G>A, the exon 4-7 region was amplified, which resulted in the expected band and a
slightly diffuse, smaller band. Sanger sequencing detected the c.546G>A change (Figure 2). However, NGS not only detected the nucleotide change, but also recognized a loss of 125 nucleotides corresponding to exon 6 (p.Thr179ProfsTer31) in small 2.3% of reads (Online Supplementary Table S4). The predicted impact of the c.546G>A mutation was discrepant and only two in silico algorithms predicted an effect on mRNA splicing (Online Supplementary Table S3) that was confirmed in vivo even though with a slight effect. To study c.4866C>T, the exon 25-31 region was amplified and sequenced, but no effect on mRNA processing was seen, as was predicted by in sil- ico tools. Both nucleotides were identified in heterozygous state, indicating the presence of both alleles. Only off- spring carrying the c.546G>A mutation (1 of 3 children) had lower levels of VWF:Ag and VWF:RCo compared to
592
haematologica | 2019; 104(3)