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| 2. |
- Manderstedt, Eric, et al.
(author)
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Genetic variation of the blood coagulation regulator tissue factor pathway inhibitor and venous thromboembolism among middle-aged and older adults: A population-based cohort study
- 2022
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In: Research and practice in thrombosis and haemostasis. - : Elsevier BV. - 2475-0379. ; 6:7
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Journal article (peer-reviewed)abstract
- Background: Tissue factor is the main initiator of blood coagulation, and tissue factor pathway inhibitor (TFPI) is the primary inhibitor of the initiation of blood coagulation. The genetic variation of TFPI and the relation to venous thromboembolism (VTE), that is, venous thrombosis and pulmonary embolism, remains to be clarified. This exome sequencing study aimed to determine the molecular epidemiology of the TFPI gene and the relation to VTE in a large population-based cohort of middle-aged and older adults. Methods: The exomes of TFPI were analyzed for variants in 28,794 subjects without previous VTE (born 1923–1950, 60% women), who participated in the Malmö Diet and Cancer Study (1991–1996). Patients were followed until the first event of VTE, death, or 2018. Qualifying variants were defined as loss-of-function or nonbenign (PolyPhen-2) missense variants with minor allele frequency less than 0.1%. Results: No common variant was associated with VTE. Nine rare variants (two loss-of-function and seven nonbenign missense) were classified as qualifying and included in collapsing analysis. Prevalence of qualifying variants was 0.09%. Five individuals with VTE compared to 17 individuals without VTE carried one qualifying variant. Cox multivariate regression analysis adjusted for age, sex, body mass index, systolic blood pressure, smoking and alcohol consumption, rs6025, rs1799963, and ancestry showed a hazard ratio of 2.9 (95% CI, 1.2–7.1) for rare qualifying variants. Conclusion: Rare qualifying TFPI variants were associated with VTE, suggesting that rare variants in TFPI contribute to the development of VTE. The qualifying TFPI gene variants were very rare, suggesting a constrained gene. © 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).
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| 3. |
- Manderstedt, Eric, et al.
(author)
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Detection of F8 int22h inversions using digital droplet PCR and mile-post assays
- 2020
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In: Journal of Thrombosis and Haemostasis. - : Wiley-Blackwell. - 1538-7933 .- 1538-7836. ; 8:5, s. 1039-1049
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Journal article (peer-reviewed)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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| 4. |
- Manderstedt, Eric, et al.
(author)
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Detection of mosaics in hemophilia A by deep Ion Torrent sequencing and droplet digital PCR
- 2020
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In: Research and practice in thrombosis and haemostasis. - : Wiley. - 2475-0379. ; 4:7, s. 1121-1130
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Journal article (peer-reviewed)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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| 6. |
- Halldén, Christer, et al.
(author)
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Origin of Swedish hemophilia A mutations
- 2012
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In: Journal of Thrombosis and Haemostasis. - : Elsevier BV. - 1538-7933 .- 1538-7836. ; 10:12, s. 2503-2511
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Journal article (peer-reviewed)abstract
- Background: Hemophilia A (HA) has a high level of variation within the disease class, with more than 1000 mutations being listed in the HAMSTeRS database. At the same time a number of F8 mutations are present in specific populations at high frequencies. Objectives: The simultaneous presence of large numbers of rare mutations and a small number of high-frequency mutations raises questions about the origins of HA mutations. The present study was aimed at describing the origins of HA mutations in the complete Swedish population. The primary issue was to determine what proportion of identical mutations are identical by descent (IBD) and what proportion are attributable to recurrent mutation events. The age of IBD mutations was also determined. Patients/Methods: In Sweden, the care of HA is centralized, and the Swedish HA population consists of 750 patients from > 300 families (35% severe, 15% moderate, and 50% mild). Identical haplotypes were defined by single-nucleotide polymorphism and microsatellite haplotyping, and the ages of the mutations were estimated with estiage. Results: Among 212 presumably unrelated patients with substitution mutations, 97 (46%) had mutations in common with other patients. Haplotyping of the 97 patients showed that 47 had IBD mutations (22%) with estimated ages of between two and 35 generations. The frequency of mild disease increased with an increasing number of patients sharing the mutations. Conclusions: A majority of the IBD mutations are mild and have age estimates of a few hundred years, but some could date back to the Middle Ages.
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| 7. |
- Halldén, Christer, et al.
(author)
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Origin of Swedish hemophilia B mutations
- 2013
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In: Journal of Thrombosis and Haemostasis. - : Elsevier BV. - 1538-7933 .- 1538-7836. ; 11:11, s. 2001-2008
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Journal article (peer-reviewed)abstract
- Background More than 1100 mutations that cause hemophilia B (HB) have been identified. At the same time, specific F9 mutations are present at high frequencies in certain populations, which raise questions about the origin of HB mutations. ObjectivesTo describe the mutation spectrum of all HB families in Sweden and investigate if mutations appearing in several families are due to independent recurrent mutations (RMs) or to a common mutation event (i.e. are identical by descent (IBD)). Patients/MethodsThe registered Swedish HB population consists of patients from 86 families. Mutations were identified by resequencing and identical haplotypes were defined using 74 markers and a control population of 285 individuals. The ages of IBD mutations were estimated using ESTIAGE. ResultsOut of 77 presumably unrelated patients with substitution mutations, 47 patients (61%) had mutations in common with other patients. Haplotyping of the 47 patients showed that 24 patients had IBD mutations (51%) with estimated ages of between two and 23 generations. A majority of these patients had mild disease. Eight of the 15 mutations observed in more than one family were C>T transitions in CpG sites and all eight were RMs. ConclusionsThe association of IBD mutations with a mild phenotype is similar to what has been previously observed in hemophilia A. Noteworthy features of the mutations that are common to more than one family are the equal proportions of patients with RM and IBD mutations and the correlation between the occurrence of RMs and C>T transitions at CpG sites.
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| 8. |
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| 9. |
- Halldén, Christer, 1957-, et al.
(author)
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Investigation of disease-associated factors in haemophilia A patients without detectable mutations
- 2012
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In: Haemophilia. - : Blackwell. - 1351-8216 .- 1365-2516. ; 18:3, s. e132-e137
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Journal article (peer-reviewed)abstract
- To investigate disease causing mechanism in haemophilia A patients without detectable mutation. Screening for F8 mutations in 307 haemophilia A patients using: re-sequencing and inversion PCR, reverse transcription (RT-PCR) of mRNA, MLPA analysis, haplotyping using SNP and microsatellite markers. No F8 mutations were detected in 9 of the 307 patients (2.9%) using re-sequencing and inversion PCR. MLPA analysis detected duplication in exon 6 in one patient and RT-PCR showed no products for different regions of mRNA in four other patients, indicating failed transcription. No obvious associations were observed between the phenotypes of the nine patients, their F8 haplotypes and the putative mutations detected. The mutation-positive patients carrying the same haplotypes as the mutation-negative patients show a multitude of different mutations, emphasizing the lack of associations at the haplotype level. VWF mutation screening and factor V measurements ruled out type 2N VWD and combined factor V and VIII deficiency respectively. To further investigate a possible role for FVIII interacting factors the haplotypes/diplotypes of F2, F9, F10 and VWF were compared. The nine patients had no specific haplotype/diplotype combination in common that can explain disease. Duplications and faulty transcription contribute to the mutational spectrum of haemophilia A patients where conventional mutation screening fail to identify mutations.
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| 10. |
- Halldén, Christer, et al.
(author)
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Investigation of disease-associated factors in haemophilia A patients without detectable mutations
- 2012
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In: Haemophilia. - : Wiley-Blackwell Publishing Ltd. - 1351-8216 .- 1365-2516. ; 18:3, s. e132-e137
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Journal article (peer-reviewed)abstract
- To investigate disease causing mechanism in haemophilia A patients without detectable mutation. Screening for F8 mutations in 307 haemophilia A patients using: re-sequencing and inversion PCR, reverse transcription (RT-PCR) of mRNA, MLPA analysis, haplotyping using SNP and microsatellite markers. No F8 mutations were detected in 9 of the 307 patients (2.9%) using re-sequencing and inversion PCR. MLPA analysis detected duplication in exon 6 in one patient and RT-PCR showed no products for different regions of mRNA in four other patients, indicating failed transcription. No obvious associations were observed between the phenotypes of the nine patients, their F8 haplotypes and the putative mutations detected. The mutation-positive patients carrying the same haplotypes as the mutation-negative patients show a multitude of different mutations, emphasizing the lack of associations at the haplotype level. VWF mutation screening and factor V measurements ruled out type 2N VWD and combined factor V and VIII deficiencyrespectively. To further investigate a possible role for FVIII interacting factors the haplotypes/diplotypes of F2, F9, F10 and VWF were compared. The nine patients had no specific haplotype/diplotype combination in common that can explain disease. Duplications and faulty transcription contribute to the mutational spectrum of haemophilia A patients where conventional mutation screening fail to identify mutations.
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