First application of MLPA method in severe von Willebrand disease. Confirmation of a new large<i>VWF</i>gene deletion and identification of heterozygous carriers

Cabrera, Noelia; Casaña, Pilar; Cid, Ana Rosa; Moret, A.; Moreno, Miguel Eduardo Torres; Palomo, Ángeles; Aznar, José A.

doi:10.1111/j.1365-2141.2010.08400.x

Cited by 16 publications

(15 citation statements)

References 8 publications

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“…VWF of these patients was entirely sequenced by the Sanger method and a mutation was detected in only 2 cases; there was no detectable mutation in the remaining 9 cases. Multiplex ligationdependent probe amplification (MLPA) 18 was applied to all these 9 cases and only patient VW0057 showed an abnormal MLPA pattern, revealing a heterozygous deletion of exon 15. The point mutations identified by Sanger sequencing and not by NGS (false-negative mutations) were a substitution in patient VW0141 (c.4120C>T) and the duplication of an amino acid (c.4678_4680dup, p.D1560dup) in patient VW0078.…”

Section: Resultsmentioning

confidence: 99%

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

Corrales¹,

Catarino²,

Ayats³

et al. 2012

Haematologica

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© 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...

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Section: Resultsmentioning

confidence: 99%

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

Corrales¹,

Catarino²,

Ayats³

et al. 2012

Haematologica

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“…1 kits P011-B1 and P012-B1 VWF (MRC-Holland, Amsterdam, Netherlands) were used, following the manufacturer instructions, to investigate the 52 exons of VWF gene [17]. For each reaction, we used 150 ng of patient genomic DNA.…”

Section: Mlpamentioning

confidence: 99%

“…HRM analysis was then carried out for 25 exons (number 4,6,8,9,11,12,16,17,19,20,21,23,25,30,34,35,36,38,39,40,41,44,46,48,51) in all patients. This is a mutation scanning technique that monitors the progressive change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it is denatured with marginal increases in temperature [18].…”

Section: Mlpamentioning

confidence: 99%

Molecular characterization, recombinant protein expression, and mRNA analysis of type 3 von Willebrand disease: Studies of an Italian cohort of 10 patients

et al. 2012

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Type 3 von Willebrand disease (VWD3) is characterized by unmeasurable von Willebrand factor (VWF) levels in plasma and platelets and severe but variable hemorrhagic symptoms. To identify and characterize the causal mutations, we screened 10 Italian patients with VWD3 by several techniques including Multiplex Ligation‐dependent Probe Amplification to identify large insertions and deletions, High Resolution Melting and PCR coupled with Sanger sequencing. Fourteen different mutations scattered throughout the VWF gene were identified, 10 of which were novel. As expected, most of these mutations caused null alleles: five were deletions (del exons 1–3, del exon 17, c.2157delA, c.2269delCT, and c.3940delG), three nonsense (p.Q1526X, p.E1549X, and p.C2448X) and three potential splice‐site mutations (c.658‐2A>G, c.7729+7C>T, and c.8155+1G>T). Three candidate missense mutations (p.C2184S, p.C2212R, and p.C2325S) were also identified. Missense mutations and the putative splice‐site defects were confirmed to be disease related by in vitro expression studies and mRNA analysis. None of these patients have developed alloantibodies against VWF. This study extends our previous finding that most of the mutations that we identified in VWD3 patients arise independently and are scattered throughout the entire VWF gene. Am. J. Hematol. 2012. © 2012 Wiley Periodicals, Inc.

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“…Gross gene rearrangements are believed to play a major role in the aetiology of human diseases, but tend to be under-explored because of the lack of straightforward techniques for their detection. The development of commercial MLPA assays for several coagulation-related genes has led to the identification of large deletions/duplications in patients with various coagulation disorders, including haemophilia A [17][18][19] and B [20], von Willebrand disease [21,22] and the deficiencies of antithrombin [16,[23][24][25], protein C [25] and protein S [25][26][27][28][29][30]. Although gross rearrangements of the F5 gene may also be more prevalent than previously suspected, only one of the 14 genetically unexplained FV-deficient patients investigated in our study turned out to carry a large deletion.…”

Section: Discussionmentioning

confidence: 99%

Identification of a novel large deletion in a patient with severe factor V deficiency using an in‐house F5 MLPA assay

et al. 2014

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Factor V (FV) deficiency is a rare autosomal recessive bleeding disorder caused by mutations in the F5 gene. FV-deficient patients in whom no mutation or only one mutation is found may harbour large gene rearrangements, which are not detected by conventional mutation screening strategies. The aim of this study was to develop and validate a multiplex ligation-dependent probe amplification (MLPA) assay for the detection of large deletions and duplications in the F5 gene. Twenty-two MLPA probes targeting 19 of the 25 exons and the upstream and downstream regions of the F5 gene were designed and tested in 10 normal controls, a patient with a known heterozygous deletion of F5 exons 1-7 (positive control) and 14 genetically unexplained FV-deficient patients. MLPA results were confirmed by digital PCR on a QuantStudio(™) 3D Digital PCR System. The F5-specific probes yielded a reproducible peak profile in normal controls, correctly detected the known deletion in the positive control and suggested the presence of a novel deletion of exons 9-10 in a patient with undetectable FV levels and only one identified mutation. Follow-up by chip-based digital PCR, long-range PCR and direct sequencing confirmed that this patient carried a heterozygous F5 deletion of 1823 bp extending from intron 8 to intron 10. Bioinformatics sequence analysis pinpointed repetitive elements that might have originated the deletion. In conclusion, we have developed and validated an MLPA assay for the detection of gross F5 gene rearrangements. This assay may represent a valuable tool for the molecular diagnosis of FV deficiency.

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First application of MLPA method in severe von Willebrand disease. Confirmation of a new largeVWFgene deletion and identification of heterozygous carriers

Cited by 16 publications

References 8 publications

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

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

Molecular characterization, recombinant protein expression, and mRNA analysis of type 3 von Willebrand disease: Studies of an Italian cohort of 10 patients

Identification of a novel large deletion in a patient with severe factor V deficiency using an in‐house F5 MLPA assay

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