Page 184 - 2019_03-Haematologica-web

N. Borràs et al.
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Figure 1. Analysis of the c.5664+2T>C mutation in patient UMP03. A) RT-PCR products amplified with primers located in exons 30 and 35 in leukocyte (L) and platelet (P) RNA, separated on 1% agarose gel. B) Traditional Sanger sequencing of PCR products from patient leukocytes (L2) and platelets (P), and control leuko- cytes (L). Analysis of the band 2 from patient leukocyte on agarose gel demonstrates exon 33 skipping. In platelets, only the allele without c.5664+2T>C mutation could be amplified. C) NGS of PCR products from leukocytes showed exon 33 skipping, indicated by arrows depict aberrant transcripts. However, exon 33-34 skipping was also detected, but in a really low of transcripts. D) Schematic representation of the mutation in genomic DNA and its effect on the VWF mRNA sequence. M indi- cates a 100-bp DNA ladder.
determined that 19% were transcripts without exon 13, 45% transcripts without exons 13 and 14, and an addi- tional aberrant transcript without exon 14 was detected in 8% transcripts (Online Supplementary Table S4). By both techniques, no effect was visible in platelets due to NMD.
The c.3379+1G>A mutation (intron 25) was identified in a type 1H VWD patient (UMP02). We designed two new primers to analyze the exon 22-26 region. Amplification of leukocyte cDNA yielded two bands, one with the expected size and a smaller band, whereas in platelets only the expected PCR product was observed (Online Supplementary Figure S4). Sanger analysis of leuko- cytes showed that c.3379+1G>A caused exon 25 skipping, leading to a frameshift at position 1075 and adding 88 aberrant amino acids before a PTC was encountered (p.Pro1075ValfsTer88). On NGS, however, two aberrant
transcripts were detected: the major one, which lacked exon 25, was detected in 43% of all reads, and the minor one, which resulted from activation of a cryptic donor splice site (DSS) 31 nucleotides upstream from the WT- DSS, was found in 1.4% of reads (p.Pro1117ValfsTer88) (Table 2 and Online Supplementary Table S4). In platelets, however, the mutated allele had undergone NMD, which precluded observation of aberrant transcripts.
The c.5664+2T>C mutation was identified in a type 1 VWD patient (UMP03), in trans with the p.Leu2407Pro mutation. To analyze the PSSM in intron 33, the exon 30- 35 region was amplified. The PCR product had the expect- ed size in platelets, whereas an additional smaller one was observed in leukocytes (Figure 1). Sanger sequencing of the leukocyte PCR product confirmed exon 33 skipping (p.Gly1874AlafsTer32) (Table 2). NGS detected 2 aberrant
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haematologica | 2019; 104(3)