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Targeted re-sequencing of F8, F9 and VWF : Characterization of Ion Torrent data and clinical implications for mutation screening

Manderstedt, Eric LU ; Nilsson, Rosanna ; Lind-Halldén, Christina ; Ljung, Rolf LU orcid ; Astermark, Jan LU and Halldén, Christer LU (2019) In PLoS ONE 14(4).
Abstract

Mutations are not identified in ~5% of hemophilia A and 10-35% of type 1 VWD patients. The bleeding tendency also varies among patients carrying the same causative mutation, potentially indicating variants in additional genes modifying the phenotype that cannot be identified by routine single-gene analysis. The F8, F9 and VWF genes were analyzed in parallel using an AmpliSeq strategy and Ion Torrent sequencing. Targeting all exonic positions showed an average read depth of >2000X and coverage close to 100% in 24 male patients with known disease-causing mutations. Discrimination between reference alleles and alternative/indel alleles was adequate at a 25% frequency threshold. In F8, F9 and VWF there was an absolute majority of all... (More)

Mutations are not identified in ~5% of hemophilia A and 10-35% of type 1 VWD patients. The bleeding tendency also varies among patients carrying the same causative mutation, potentially indicating variants in additional genes modifying the phenotype that cannot be identified by routine single-gene analysis. The F8, F9 and VWF genes were analyzed in parallel using an AmpliSeq strategy and Ion Torrent sequencing. Targeting all exonic positions showed an average read depth of >2000X and coverage close to 100% in 24 male patients with known disease-causing mutations. Discrimination between reference alleles and alternative/indel alleles was adequate at a 25% frequency threshold. In F8, F9 and VWF there was an absolute majority of all reference alleles at allele frequencies >95% and the average alternative allele and indel frequencies never reached above 10% and 15%, respectively. In VWF, 4-5 regions showed lower reference allele frequencies; in two regions covered by the pseudogene close to the 25% cut-off for reference alleles. All known mutations, including indels, gross deletions and substitutions, were identified. Additional VWF variants were identified in three hemophilia patients. The presence of additional mutations in 2 out of 16 (12%) randomly selected hemophilia patients indicates a potential mutational contribution that may affect the disease phenotype and counseling in these patients. Parallel identification of disease-causing mutations in all three genes not only confirms the deficiency, but differentiates phenotypic overlaps and allows for correct genetic counseling.

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author
; ; ; ; and
publishing date
type
Contribution to journal
publication status
published
subject
in
PLoS ONE
volume
14
issue
4
article number
e0216179
publisher
Public Library of Science (PLoS)
external identifiers
  • pmid:31026269
  • scopus:85064817851
ISSN
1932-6203
DOI
10.1371/journal.pone.0216179
language
English
LU publication?
no
id
d6986b1f-0892-4aea-b322-c8d667641156
date added to LUP
2019-04-29 15:50:53
date last changed
2025-10-14 10:37:02
@article{d6986b1f-0892-4aea-b322-c8d667641156,
  abstract     = {{<p>Mutations are not identified in ~5% of hemophilia A and 10-35% of type 1 VWD patients. The bleeding tendency also varies among patients carrying the same causative mutation, potentially indicating variants in additional genes modifying the phenotype that cannot be identified by routine single-gene analysis. The F8, F9 and VWF genes were analyzed in parallel using an AmpliSeq strategy and Ion Torrent sequencing. Targeting all exonic positions showed an average read depth of &gt;2000X and coverage close to 100% in 24 male patients with known disease-causing mutations. Discrimination between reference alleles and alternative/indel alleles was adequate at a 25% frequency threshold. In F8, F9 and VWF there was an absolute majority of all reference alleles at allele frequencies &gt;95% and the average alternative allele and indel frequencies never reached above 10% and 15%, respectively. In VWF, 4-5 regions showed lower reference allele frequencies; in two regions covered by the pseudogene close to the 25% cut-off for reference alleles. All known mutations, including indels, gross deletions and substitutions, were identified. Additional VWF variants were identified in three hemophilia patients. The presence of additional mutations in 2 out of 16 (12%) randomly selected hemophilia patients indicates a potential mutational contribution that may affect the disease phenotype and counseling in these patients. Parallel identification of disease-causing mutations in all three genes not only confirms the deficiency, but differentiates phenotypic overlaps and allows for correct genetic counseling.</p>}},
  author       = {{Manderstedt, Eric and Nilsson, Rosanna and Lind-Halldén, Christina and Ljung, Rolf and Astermark, Jan and Halldén, Christer}},
  issn         = {{1932-6203}},
  language     = {{eng}},
  month        = {{04}},
  number       = {{4}},
  publisher    = {{Public Library of Science (PLoS)}},
  series       = {{PLoS ONE}},
  title        = {{Targeted re-sequencing of F8, F9 and VWF : Characterization of Ion Torrent data and clinical implications for mutation screening}},
  url          = {{http://dx.doi.org/10.1371/journal.pone.0216179}},
  doi          = {{10.1371/journal.pone.0216179}},
  volume       = {{14}},
  year         = {{2019}},
}