In 40 out of 45 families, the sporadic case resulted from a mutation in the last two generations. In 82% (23/28), the carrier mothers had a de novo mutation where the X chromosome was of paternal origin in 74% (17/23). ddPCR is a potentially powerful and promising analysis for mosaicism in HA.

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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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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 polymerase chain reaction (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 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% to 40% linked molecules for loci 12 to15 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. K E Y W O R D Sfactor VIII, genetic linkage, hemophilia A, polymerase chain reaction, sequence inversion