High-throughput molecular diagnosis of von Willebrand disease by next generation sequencing methods

Abstract

© F e r r a t a S t o r t i F o u n d a t i o nregions and flanking intronic regions of VWF (~10 Kb) are included ( Figure 1A). Normalization of amplicons was carried out by gel quantification performed with ImageJ (Version 1.43u), a public domain Java image processing program (http://rsbweb.nih.gov/ij/). By taking a sample with a known concentration as the control, it was possible to extrapolate the concentration of each sample to create a normalized pool of the 47 PCR products in one tube. This pooling process produced 8 tubes and each tube was the result of simultaneous amplification of VWF of 5 patients. Polymerase chain reaction fragmentation, massively parallel sequencing and bioinformatic analysisIn NGS, the fragmentation step is critical for the success of the sequencing process and must be performed according to stringent parameters. Because the commonly used mechanical systems for genomic DNA fragmentation such as sonication are not effective for short DNA fragments (e.g., <300-500 bp), 12 conventional short PCRs were fragmented by heat incubation at 95ºC for 9 h ( Figure 1B) followed by 1 h of re-annealing at 72°C. This provided efficient DNA fragmentation that was relatively unbiased, as required for Illumina GA sequencing procedures.12 All samples were purified using the QIAquick PCR Purification Kit (Qiagen) and the size distribution of the fragments was checked using a DNA 100 chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Subsequent massively parallel sequencing and bioinformatic analysis are described in the Online Supplementary Design and Methods section. Mutation assignmentTo confirm the mutations identified by NGS and assign them to patients, the specific region including the mutation was PCRamplified and sequenced by dideoxynucleotide method, as described. 4 The sequences obtained were assembled and aligned against the consensus wild-type VWF sequence using SeqScape software (v2.7) (Applied Biosystems, Foster City, CA, USA). Results and DiscussionBased on the conclusions from a pilot study (Online Supplementary Appendix), a proof-of-concept study was performed for the simultaneous analysis by NGS of 40 VWD patients previously diagnosed and classified according to their clinical characteristics (Table 1). The mutations responsible for the disease were known in 8 of the selected patients who had been characterized by Sanger sequencing 4 and were used as controls. Patients included in this study were randomly selected from all those who met the criteria for VWD, to obtain a representative picture of the types, percentages, and variability of patients usually seen at the Hemophilia Unit.NGS technologies are economically extremely advantageous when the sequencer runs completely full which, in the case of short region sequencing (e.g., VWF), means including the maximum number of samples 14 and the preparation of one sequencing library per sample. However, library construction is one of the most expensive steps in the overall NGS process. Therefore, when t...

Rare and low-frequency variants and their association with plasma levels of fibrinogen, FVII, FVIII, and vWF

“…rs7962217 was associated with higher FVIII levels whereas rs41276738 was associated with lower levels and had an effect size similar to that of the strongest genetic predictor of FVIII levels, the O-deletion tagging SNP (rs657152). rs41276738 has been reported in patients with von Willebrand disease type 1 34,35 and type 2N, [36][37][38][39][40][41][42][43] but the association with vWF levels did not reach exome-wide significance, although its direction was consistent with the direction of effects on FVIII. The STAB2 variant rs141041254 was associated with higher plasma levels of both FVIII and vWF.…”

“…Some of the variants have been found in patients with diseases related to blood clotting, which suggests that these genes and their uncommon and rare genetic variation may play a role in a clinical phenotype. [26][27][28][32][33][34][35][36][37][38][39][40][41][42][43] The distribution of the phenotypes within our research populations were within the extremes of a clinically activity, and 2% to 297% antigen; FVIII: 14% to 500% activity; and vWF: 2% to 374% antigen). Furthermore, the magnitude of difference in the phenotype associated with the variant was mostly modest, although some were larger and were associated with a change equivalent to half the size of the estimated population mean for the phenotype of interest.…”

VWF sequence variants: a data goldmine

“…However, relatively few patients achieve deep and sustainable responses that would render them eligible for a stopping study similar to STIM or TWISTER. Milojkovic et al 8 over time. But we are left with the fact that after 14 years of imatinib therapy, less than 5% of patients have been able to durably discontinue therapy.…”

Molecular diagnosis of von Willebrand disease

“…These authors used a modified version of the NGS method previously reported by Corrales et al [20] and the reliability of this NGS approach has already been confirmed by Fidalgo and colleagues [22] on a cohort of Portuguese VWD patients. In Figure 2 a schematic representation of this NGS procedure is summarized.…”

“…Newly developed genetic methods such as NGS are becoming faster and progressively cheaper, allowing wider study of VWF mutations at a very reasonable cost [20]. NGS enables investigation of the entire VWF coding region in a short time and therefore this approach is better suited to investigate VWD types 1 and 3 variants.…”