| 1. |
- Lind-Halldén, Christina, et al.
(författare)
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Genetic variation in the syntaxin-binding protein STXBP5 in type 1 von Willebrand disease patients
- 2018
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Ingår i: Thrombosis and haemostasis. - 2567-689X. ; 118:8, s. 1382-1389
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Tidskriftsartikel (refereegranskat)abstract
- von Willebrand factor (VWF) levels in healthy individuals and in patients with type 1 von Willebrand disease (VWD) are influenced by genetic variation in several genes, for example, VWF, ABO and STXBP5. Here, we comprehensively screen for STXBP5 variants and investigate their association with type 1 VWD in Swedish patients and controls. The coding region of the STXBP5 gene was re-sequenced in 107 type 1 VWD patients and the detected variants were genotyped in the type 1 VWD population and a Swedish control population (464 individuals). The functional effects of missense alleles were predicted in silico and the pattern of genetic variation in STXBP5 was analysed. Re-sequencing of 107 type 1 VWD patients identified three missense and three synonymous variants in the coding sequence of STXBP5. The low-frequency missense variants rs144099092 (0.005) and rs148830578 (0.029) were predicted to be damaging, but were not accumulated in patients. No other rare candidate mutations were detected. STXBP5 showed a high level of linkage disequilibrium and a low overall nucleotide diversity of π = 3.2 × 10-4 indicating intolerance to variants affecting protein function. Three previously type 1 VWD-associated single nucleotide polymorphisms were located on one haplotype that showed an increased frequency in patients versus controls. No differences in messenger ribonucleic acid abundance among haplotypes could be found using Genotype-Tissue Expression project data. In conclusion, a haplotype containing the STXBP5 Asn436Ser (rs1039084) mutation is associated with type 1 VWD and no rare STXBP5 mutations contribute to type 1 VWD in the Swedish population.
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| 2. |
- Lind-Halldén, Christina, et al.
(författare)
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Genetic variation in the syntaxin-binding protein STXBP5 in type 1 von Willebrand disease patients
- 2018
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Ingår i: Thrombosis and Haemostasis. - : Georg Thieme Verlag. - 2567-689X .- 0340-6245. ; 118:8, s. 1382-1389
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Tidskriftsartikel (refereegranskat)abstract
- von Willebrand factor (VWF) levels in healthy individuals and in patients with type 1 von Willebrand disease (VWD) are influenced by genetic variation in several genes, for example, VWF, ABO and STXBP5. Here, we comprehensively screen for STXBP5 variants and investigate their association with type 1 VWD in Swedish patients and controls. The coding region of the STXBP5 gene was re-sequenced in 107 type 1 VWD patients and the detected variants were genotyped in the type 1 VWD population and a Swedish control population (464 individuals). The functional effects of missense alleles were predicted in silico and the pattern of genetic variation in STXBP5 was analysed. Re-sequencing of 107 type 1 VWD patients identified three missense and three synonymous variants in the coding sequence of STXBP5. The low-frequency missense variants rs144099092 (0.005) and rs148830578 (0.029) were predicted to be damaging, but were not accumulated in patients. No other rare candidate mutations were detected. STXBP5 showed a high level of linkage disequilibrium and a low overall nucleotide diversity of π = 3.2 × 10-4 indicating intolerance to variants affecting protein function. Three previously type 1 VWD-associated single nucleotide polymorphisms were located on one haplotype that showed an increased frequency in patients versus controls. No differences in messenger ribonucleic acid abundance among haplotypes could be found using Genotype-Tissue Expression project data. In conclusion, a haplotype containing the STXBP5 Asn436Ser (rs1039084) mutation is associated with type 1 VWD and no rare STXBP5 mutations contribute to type 1 VWD in the Swedish population.
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| 3. |
- Cartwright, Ashley, et al.
(författare)
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Characterization of large in-frame von Willebrand factor deletions highlights differing pathogenic mechanisms
- 2020
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Ingår i: Blood Advances. - 2473-9529 .- 2473-9537. ; 4:13, s. 2979-2990
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Tidskriftsartikel (refereegranskat)abstract
- Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion.
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| 4. |
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| 5. |
- Manderstedt, Eric, et al.
(författare)
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Detection of F8 int22h inversions using digital droplet PCR and mile-post assays
- 2020
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Ingår i: Journal of Thrombosis and Haemostasis. - : Wiley-Blackwell. - 1538-7933 .- 1538-7836. ; 8:5, s. 1039-1049
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Inversions involving intron 22 (Inv22) of F8 are detected in approximately 45% of all severe hemophilia A patients. Diagnosis is complicated by the large size of the ~9.5 kb int22h repeated sequence which generates the inversions. Methods such as long-range PCR and inverse-shifting PCR are currently used diagnostically, but suffer from low PCR efficiencies and are difficult to standardize.OBJECTIVES: To design and validate a sensitive and robust assay for the detection of F8 int22h inversions.METHODS: Digital droplet PCR using mile-post assays was used to investigate archival DNA samples.RESULTS: The detection of linkage as a function of physical distance between loci was investigated using an anchor locus and mile-post loci located at 1, 6, 12 and 15 kb distances from the anchor locus. The proportion of linked molecules decreased with increasing distance between loci and showed 30-40% linked molecules for loci 12-15 kb apart. Mile-post assays specific for wild type and Inv22 type 1 and 2 chromosomes were then designed and optimized. All three assays showed high specificities and sensitivities, with coefficients of variation < 5% for all assays. Analysis of 106 patients and 20 carrier mothers showed complete concordance with previously known mutation status. The analysis demonstrated the robustness of the assays versus input DNA concentration (6 ng and higher) and level of fragmentation.CONCLUSIONS: Digital droplet PCR and mile-post assays can be used to detect F8 int22h inversions. The assay systems are technically simple to perform, highly efficient and robust.
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| 6. |
- Manderstedt, Eric, et al.
(författare)
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Detection of mosaics in hemophilia A by deep Ion Torrent sequencing and droplet digital PCR
- 2020
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Ingår i: Research and practice in thrombosis and haemostasis. - : Wiley. - 2475-0379. ; 4:7, s. 1121-1130
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Tidskriftsartikel (refereegranskat)abstract
- Background: The occurrence of mosaicism in hemophilia A (HA) has been investigated in several studies using different detection methods. Objectives: To characterize and compare the ability of AmpliSeq/Ion Torrent sequencing and droplet digital polymerase chain reaction (ddPCR) for mosaic detection in HA. Methods: Ion Torrent sequencing and ddPCR were used to analyze 20 healthy males and 16 mothers of sporadic HA patients. Results: An error-rate map over all coding positions and all positions reported as mutated in the F8-specific mutation database was produced. The sequencing produced a mean read depth of >1500X where >97% of positions were covered by >100 reads. Higher error frequencies were observed in positions with A or T as reference allele and in positions surrounded on both sides with C or G. Seventeen of 9319 positions had a mean substitution error frequency >1%. The ability to identify low-level mosaicism was determined primarily by read depth and error rate of each specific position. Limit of detection (LOD) was <1% for 97% of positions with substitutions and 90% of indel positions. The positions with LOD >1% require repeated testing and mononucleotide repeats with more than four repeat units need an alternative analysis strategy. Mosaicism was detected in 1 of 16 mothers and confirmed using ddPCR. Conclusions: Deep sequencing using an AmpliSeq/Ion Torrent strategy allows for simultaneous identification of disease-causing mutations in patients and mosaicism in mothers. ddPCR has high sensitivity but is hampered by the need for mutationspecific design.
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| 7. |
- Manderstedt, Eric, et al.
(författare)
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Droplet digital PCR and mile-post analysis for the detection of F8 int1h inversions
- 2021
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Ingår i: Journal of Thrombosis and Haemostasis. - : Wiley-Blackwell. - 1538-7933 .- 1538-7836. ; 19:3, s. 732-737
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: F8 int1h inversions (Inv1) are detected in 1-2% of severe hemophilia A (HA) patients. Long-range polymerase chain reaction (PCR) and inverse-shifting PCR have been used to diagnose these inversions.OBJECTIVES: To design and validate a sensitive and robust assay for detection of F8 Inv1 inversions.METHODS: Archival DNA samples were investigated using mile-post assays and droplet digital PCR.RESULTS: Mile-post assays for Inv1 showing high specificities and sensitivities were designed and optimized. Analysis of four patients, two carrier mothers and 40 healthy controls showed concordance with known mutation status with one exception. One patient had a duplication involving exons 2-22 of the F8 gene instead of an Inv1 mutation. DNA mixtures with different proportions of wild type and Inv1 DNA correlated well with the observed relative linkage for both wild type and Inv1 assays and estimated the limit of detection of these assays to 2% of the rare chromosome.CONCLUSIONS: The mile-post strategy has several inherent control systems. The absolute counting of target molecules by both assays enables determination of template quantity, detection of copy number variants and rare variants occurring in primer and probe annealing sites and estimation of DNA integrity through the observed linkage. The presented Inv1 mile-post analysis offers sensitive and robust detection and quantification of the F8 int1h inversions and other rearrangements involving intron 1 in patients and their mothers.
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| 8. |
- Mufti, Ahmad H, et al.
(författare)
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The common VWF single nucleotide variants c.2365A>G and c.2385T>C modify VWF biosynthesis and clearance
- 2018
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Ingår i: Blood Advances. - 2473-9529 .- 2473-9537. ; 2:13, s. 1585-1594
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Tidskriftsartikel (refereegranskat)abstract
- Plasma levels of von Willebrand factor (VWF) vary considerably in the general population and this variation has been linked to several genetic and environmental factors. Genetic factors include 2 common single nucleotide variants (SNVs) located in VWF, rs1063856 (c.2365A>G) and rs1063857 (c.2385T>C), although to date the mechanistic basis for their association with VWF level is unknown. Using genotypic/phenotypic information from a European healthy control population, in vitro analyses of recombinant VWF expressing both SNVs, and in vivo murine models, this study determined the precise nature of their association with VWF level and investigated the mechanism(s) involved. Possession of either SNV corresponded with a significant increase in plasma VWF in healthy controls (P < .0001). In vitro expression confirmed this observation and highlighted an independent effect for each SNV (P < .0001 and P < .01, respectively), despite close proximity and strong linkage disequilibrium between them both. The influence of c.2365A>G on VWF levels was also confirmed in vivo. This increase in VWF protein corresponded to an increase in VWF messenger RNA (mRNA) resulting, in part, from prolonged mRNA half-life. In addition, coinheritance of both SNVs was associated with a lower VWF propeptide-to-VWF antigen ratio in healthy controls (P < .05) and a longer VWF half-life in VWF knockout mice (P < .0001). Both SNVs therefore directly increase VWF plasma levels through a combined influence on VWF biosynthesis and clearance, and may have an impact on disease phenotype in both hemostatic and thrombotic disorders.
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| 9. |
- Svahn, Ola, 1970-, et al.
(författare)
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Interkalibrerad läkemedelsanalys 2017 : ett samarbetsprojekt för ökad analyskvalité
- 2017
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Läkemedelsverket gav i september 2015 ut en rapport med titeln ”Miljöindikatorer inom ramen för nationella läkemedelsstrategin (NLS)”. Rapporten indikerar bland annat stora kvalitetsbrister i tidigare utförda analyser av läkemedel i miljön under åren 2002-2013, men betonar också vikten av att samordna framtida nationella läkemedelsanalyser för att bättre använda våra offentliga resurser. I rapporten skriver man följande: ”Allra högst prioriterad ansåg arbetsgruppen att indikatorn ”mäta halter av läkemedelssubstanser i miljö” vara. Detta beroende på att det utöver att det är av stor vikt att följa utvecklingen av läkemedelsrester i miljön över tid för att utvärdera effekten av genomförda insatser, så ansåg arbetsgruppen att det finns stora möjligheter att optimera användningen av de offentliga resurserna genom en bättre samordning av mätningar i miljön. Många mätningar har gjorts historiskt av olika offentliga aktörer utan någon samordning.” Mot bakgrund av detta, och som svar på ett nationellt behov, åtog sig Högskolan Kristianstad (HKR) genom Ola Svahn och Erland Björklund, båda verksamma vid MoLab, Krinova Incubator and Science Park i Kristianstad, att leda denna interkalibreringsstudie för läkemedelsanalys i miljö-vattenprover av olika typ. Arbetet har skett på uppdrag av Havs- och Vattenmyndigheten som finansierat arbetet via bidrag ur havs- och vattenmiljöanslaget och i samarbete med fyra andra analyslaboratorier vid Umeå Universitet (UU), Sveriges Lantbruksuniversitet Uppsala (SLU), Svenska miljöinstitutet Stockholm (IVL) samt Aarhus Universitet Danmark (AU). Total deltog därmed 5 laboratorier, alla med tidigare erfarenhet av läkemedelsanalys i miljöprover.
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| 10. |
- Zöller, Bengt, et al.
(författare)
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Genetic risk factors for venous thromboembolism
- 2020
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Ingår i: Expert Review of Hematology. - 1747-4086 .- 1747-4094. ; 13:9, s. 971-981
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: Venous thromboembolism (VTE) is a complex disease that aggregates in families. Both acquired and genetic risk factors are important. Proper recognition and management of high-risk individuals are important.AREAS COVERED: The genetic risk factors for VTE, the clinical consequences, and future perspectives are summarized. Classical thrombophilia i.e. factor V Leiden (rs6025), the prothrombin G20210A mutation (rs1799963), deficiencies of antithrombin, protein C, and protein S and the recent findings from genome wide association studies (GWAS), transcriptome-wide association studies (TWAS), genetic risk score (GRS), VTE candidate genes, expression studies, animal studies, studies using next generation sequencing, pathway analysis, and clinical implications are discussed.EXPERT OPINION: Screening of inherited thrombophilia should be performed in special cases. Identification of strong risk variants might affect the management. The increasing number of genetic risk variants is likely to change management of VTE.
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