Background In a small group of typical hemophilia A (HA) patients no mutations in the F8 coding sequence (cDNA) could be found. In the current study, we performed a systematic screening of genetic and non‐genetic parameters associated with reduced FVIII:C levels in a group of mostly mild HA (only one moderate) patients with no detectable mutations in F8 cDNA. Methods We determined FVIII and VWF activity and antigen levels and performed VWF‐FVIII binding (VWF:FVIIIB) and VWF‐collagen binding assays (VWF:CB) as well as VWF multimer analysis. VWF was completely sequenced to exclude mutations. The F8 locus, including the introns, was sequenced using overlapping long‐range PCRs (LR‐PCRs) combined with a next generation sequencing (NGS) approach. Moreover, the F8 mRNA was analyzed quantitatively and qualitatively by real‐time PCR (qRT) and overlapping reverse transcription (RT) PCRs, respectively. Results All VWF tests were normal. The LR‐PCRs demonstrated the integrity of the F8 locus. Eight unique polymorphisms were found in the patients, with two being recurrent. Furthermore, RT‐PCRs analysis confirmed that two of the unique variants create detectable new cryptic splice sites in the patients that result in the introduction of intronic DNA sequences into the mRNA and create premature stop codons. Conclusion By systematically excluding all possible causes of HA, we could with great certainty conclude that deep intronic mutations in F8, although rare, cause abnormal mRNA splicing, leading to mild HA.

About one-third of patients with non-severe haemophilia A (HA) show a discrepancy of factor (F)VIII activity (FVIII:C) measured by one-stage (FVIII:C_1st), two-stage assays or the chromogenic method (FVIII:C_chr). The aim of the study was to characterise the mutation profile in patients with FVIII:C assay discrepancies. FVIII:C discrepancy was considered significant if the calculated ratio between FVIII:C_1st and FVIII:C_chr was ≤ 0.6. In 16 patients FVIII:C_1st was higher than FVIII:C_chr. The reverse phenomenon was observed in 83 patients. Genetic analysis revealed 23 different missense mutations of which 17 were novel. Most mutations, exhibiting a higher FVIII:C_1st were localised in the A1-A2-A3 interface. The majority of mutations associated with FVIII:C_chr>FVIII:C_1st discrepancy were located close to or within the thrombin cleavage sites, FIX or vWF binding sites. Our data show a correlation between FVIII:C and thrombin generation testing with a clear differentiation between patients with haemophilia and normal controls. However, in the subgroup of FVIII:C_1st>FVIII:C_chr discrepancy, the endogenous thrombin potential and peak thrombin parameters were similar to non-discrepant haemophilia patients, while in the inverse discrepancy FVIII:C_chr>FVIII:C_1st, these variables showed values close to that of the normal control group. Certain hereditary F8 mis-sense mutations cause discrepancy in FVIII:C as measured with different assays. This can lead to failure in diagnosing HA or incorrectly classifying the severity of the disease. Therefore, we recommend that initial diagnosis of non-severe HA phenotypes should be based on results of both FVIII:C_1st and FVIII:C_chr assays.

Molecular genetic analysis is widely applied in inherited bleeding disorders. The outcome of genetic analysis allows genetic counselling in affected families and helps to find a link between the genotype and phenotype. Genetic analysis in hemophilia A (HA) has tremendously improved in the past decades. Many new techniques and modifications as well as analysis software have become available, which has enabled genetic analysis and interpretation of data to become faster and more accurate. The advances in mutation detection strategies facilitate the identification of the causal mutation in up to 97% of patients with HA. This review discusses the milestones in genetic analysis of HA and highlights the importance of identification of the causative mutations for genetic counseling and particularly for the interpretation of the clinical presentation of HA patients. ^* This article is dedicated to Dr. Hans-Hermann Brackmann in recognition of his pioneering work for hemophilia patients.

Summary: The aim of molecular genetic analysis in families with haemophilia is to identify the causative mutation in an affected male as this provides valuable information for the patient and his relatives. For the patient, mutation identification may highlight inhibitor development risk or discrepancy between different factor VIII assays. For female relatives, knowledge of the familial mutation can facilitate carrier status determination and prenatal diagnosis. Recent advances in understanding mutations responsible for haemophilia and methods for their detection are presented. For reporting of such mutations, participation in external quality assessment ensures that essential patient and mutation details are routinely included and that pertinent information is incorporated in the interpretation.