| 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. |
- Manderstedt, Eric, et al.
(författare)
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Classic Thrombophilias and Thrombotic Risk Among Middle-Aged and Older Adults : A Population-Based Cohort Study
- 2022
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Ingår i: Journal of the American Heart Association. - : Wiley-Blackwell Publishing Ltd. - 2047-9980. ; 11:4
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Five classic thrombophilias have been recognized: factor V Leiden (rs6025), the prothrombin G20210A variant (rs1799963), and protein C, protein S, and antithrombin deficiencies. This study aimed to determine the thrombotic risk of classic thrombophilias in a cohort of middle-aged and older adults. METHODS AND RESULTS: Factor V Leiden, prothrombin G20210A and protein-coding variants in the PROC (protein C), PROS1 (protein S), and SERPINC1 (antithrombin) anticoagulant genes were determined in 29 387 subjects (born 1923–1950, 60% women) who participated in the Malmö Diet and Cancer study (1991–1996). The Human Gene Mutation Database was used to define 68 disease-causing mutations. Patients were followed up from baseline until the first event of venous thromboembolism (VTE), death, or Dec 31, 2018. Carriership (n=908, 3.1%) for disease-causing mutations in the PROC, PROS1, and SERPINC1 genes was associated with incident VTE: Hazard ratio (HR) was 1.6 (95% CI, 1.3–1.9). Variants not in Human Gene Mutation Database were not linked to VTE (HR, 1.1; 95% CI, 0.8–1.5). Heterozygosity for rs6025 and rs1799963 was associated with incident VTE: HR, 1.8 (95% CI, 1.6–2.0) and HR, 1.6 (95% CI, 1.3–2.0), respectively. The HR for carrying 1 classical thrombophilia variant was 1.7 (95% CI, 1.6–1.9). HR was 3.9 (95% CI, 3.1–5.0) for carriers of ≥2 thrombophilia variants. CONCLUSIONS: The 5 classic thrombophilias are associated with a dose-graded risk of VTE in middle-aged and older adults. Disease-causing variants in the PROC, PROS1, and SERPINC1 genes were more common than the rs1799963 variant but the conferred genetic risk was comparable with the rs6025 and rs1799963 variants.
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| 4. |
- Manderstedt, Eric, et al.
(författare)
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Common and rare vriants in genes associated with von Willebrand factor level variation : no accumulation of rare variants in Swedish von Willebrand disease patients
- 2020
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Ingår i: TH open : companion journal to thrombosis and haemostasis. - 2512-9465. ; 4:4, s. 322-331
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Tidskriftsartikel (refereegranskat)abstract
- Genome-wide association studies (GWASs) have identified genes that affect plasma von Willebrand factor (VWF) levels. ABO showed a strong effect, whereas smaller effects were seen for VWF , STXBP5 , STAB2 , SCARA5 , STX2 , TC2N , and CLEC4M . This study screened comprehensively for both common and rare variants in these eight genes by resequencing their coding sequences in 104 Swedish von Willebrand disease (VWD) patients. The common variants previously associated with the VWF level were all accumulated in the VWD patients compared to three control populations. The strongest effect was detected for blood group O coded for by the ABO gene (71 vs. 38% of genotypes). The other seven VWF level associated alleles were enriched in the VWD population compared to control populations, but the differences were small and not significant. The sequencing detected a total of 146 variants in the eight genes. Excluding 70 variants in VWF , 76 variants remained. Of the 76 variants, 54 had allele frequencies > 0.5% and have therefore been investigated for their association with the VWF level in previous GWAS. The remaining 22 variants with frequencies < 0.5% are less likely to have been evaluated previously. PolyPhen2 classified 3 out of the 22 variants as probably or possibly damaging (two in STAB2 and one in STX2 ); the others were either synonymous or benign. No accumulation of low frequency (0.05-0.5%) or rare variants (<0.05%) in the VWD population compared to the gnomAD (Genome Aggregation Database) population was detected. Thus, rare variants in these genes do not contribute to the low VWF levels observed in VWD patients.
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| 5. |
- Manderstedt, Eric, et al.
(författare)
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Common and rare vriants in genes associated with von Willebrand factor level variation : no accumulation of rare variants in Swedish von Willebrand disease patients
- 2020
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Ingår i: TH open : companion journal to thrombosis and haemostasis. - : Georg Thieme Verlag KG. - 2512-9465. ; 4:4, s. 322-331
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Tidskriftsartikel (refereegranskat)abstract
- Genome-wide association studies (GWASs) have identified genes that affect plasma von Willebrand factor (VWF) levels. ABO showed a strong effect, whereas smaller effects were seen for VWF , STXBP5 , STAB2 , SCARA5 , STX2 , TC2N , and CLEC4M . This study screened comprehensively for both common and rare variants in these eight genes by resequencing their coding sequences in 104 Swedish von Willebrand disease (VWD) patients. The common variants previously associated with the VWF level were all accumulated in the VWD patients compared to three control populations. The strongest effect was detected for blood group O coded for by the ABO gene (71 vs. 38% of genotypes). The other seven VWF level associated alleles were enriched in the VWD population compared to control populations, but the differences were small and not significant. The sequencing detected a total of 146 variants in the eight genes. Excluding 70 variants in VWF , 76 variants remained. Of the 76 variants, 54 had allele frequencies > 0.5% and have therefore been investigated for their association with the VWF level in previous GWAS. The remaining 22 variants with frequencies < 0.5% are less likely to have been evaluated previously. PolyPhen2 classified 3 out of the 22 variants as probably or possibly damaging (two in STAB2 and one in STX2 ); the others were either synonymous or benign. No accumulation of low frequency (0.05-0.5%) or rare variants (<0.05%) in the VWD population compared to the gnomAD (Genome Aggregation Database) population was detected. Thus, rare variants in these genes do not contribute to the low VWF levels observed in VWD patients.
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| 6. |
- 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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| 7. |
- 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 Publishing Ltd. - 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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| 8. |
- 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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| 9. |
- 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. - : Elsevier BV. - 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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| 10. |
- 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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