Introduction Familial multiple coagulation factor deficiencies (FMCFDs) are a group of rare inherited
hemostatic disorders characterized by a simultaneous reduction of plasma activity
of at least two coagulation factors. The detection of multiple deficiencies was based
on systematic screening of coagulation factors in patients exhibiting atypical bleeding
patterns, in pre-surgery management, or familial investigations using Sanger Sequencing.
The introduction of Next Generation Sequencing (NGS) in routine molecular genetic
testing has significantly increased the identification of multiple genetic alterations,
albeit often as incidental findings, posing challenges.
Method Approximately, 7500 index patients (IPs) with known deficiencies in one or more than
one coagulation factors have been screened by Sanger Sequencing and Multiplex Ligand
Probe Analysis, with around 3000 IPs analyzed by NGS and Copy Number Variant Analysis.
Results The use of NGS has led to a twofold rise in the detection rate of multiple coagulation
factor deficiencies (82 FMCFD IPs in 13 years – 1,1% against 54 FMCFD IPs in 4,5 years
– 1,9%), particularly in FMCFD IPS with variants present in at least two different
genes. Within the NGS-based cohort, 106 genetic variants were identified, with 23%
classified as variant of uncertain significance (VUS) and 33% of them were incidental
findings. Notably, male severe hemophilic IPs show the incidental finding in the additional
affected gene – due to the dominant clinical phenotype of F8 or F9 defect. In contrast,
incidentals findings in hemophilia carriers were predominantly found in the F8- and
F9 gene – explainable with the X-linked inheritance. The highest rate of incidental
findings was observed in IPs with genetic variants in one pro- and one anticoagulant
factor, where 50% of the variants would have remained undetected without NGS. Remarkably,
17% of all genetic variants in the entire NGS cohort affected the VWF gene, and 63%
of IPs with defects in one pro- and one anticoagulant factor showed involvement of
the VWF gene. Interestingly, in one family with VWD type 3, a variant in the second
gene (SERPINC1) completely mitigated the phenotype of VWS [1]
[2].
Conclusion The implication of NGS in molecular genetic diagnostics has nearly doubled the detection
rate of FMCFDs with a predominance of VWD. NGS not only supports the detection of
carriers for hemophilia, but also enables the identification of VWD in IPs with no
bleeding symptoms due to a secondary defect in an anti-coagulant factor. Additionally,
family segregation analysis and expression studies will help to improve the assessment
of VUS pathogenicity. Overall, NGS supports tailored treatment approaches of FMCFD
IPs and facilitates effective family counselling.
