Date Published: April 26, 2019
Publisher: Public Library of Science
Author(s): Eric Manderstedt, Rosanna Nilsson, Christina Lind-Halldén, Rolf Ljung, Jan Astermark, Christer Halldén, Pablo Garcia de Frutos.
Mutations are not identified in ~5% of hemophilia A and 10–35% of type 1 VWD patients. The bleeding tendency also varies among patients carrying the same causative mutation, potentially indicating variants in additional genes modifying the phenotype that cannot be identified by routine single-gene analysis. The F8, F9 and VWF genes were analyzed in parallel using an AmpliSeq strategy and Ion Torrent sequencing. Targeting all exonic positions showed an average read depth of >2000X and coverage close to 100% in 24 male patients with known disease-causing mutations. Discrimination between reference alleles and alternative/indel alleles was adequate at a 25% frequency threshold. In F8, F9 and VWF there was an absolute majority of all reference alleles at allele frequencies >95% and the average alternative allele and indel frequencies never reached above 10% and 15%, respectively. In VWF, 4–5 regions showed lower reference allele frequencies; in two regions covered by the pseudogene close to the 25% cut-off for reference alleles. All known mutations, including indels, gross deletions and substitutions, were identified. Additional VWF variants were identified in three hemophilia patients. The presence of additional mutations in 2 out of 16 (12%) randomly selected hemophilia patients indicates a potential mutational contribution that may affect the disease phenotype and counseling in these patients. Parallel identification of disease-causing mutations in all three genes not only confirms the deficiency, but differentiates phenotypic overlaps and allows for correct genetic counseling.
Inherited bleeding disorders affect an estimated 7.5 million individuals worldwide . The common inherited bleeding disorders, von Willebrand disease (VWD), hemophilia A (HA) and hemophilia B (HB), account for 95–97% of such patients . VWD is the most common inherited autosomal bleeding disorder, affecting both genders. Mutations in the VWF gene result in quantitative or qualitative deficiencies of von Willebrand factor (VWF). The estimated prevalence of subjects with significant bleeding symptoms is 1 in 1000 . More than 700 mutations have been associated with VWD (https://grenada.lumc.nl/LOVD2/VWF/home.php?select_db=VWF). HA and HB are X-linked recessive disorders caused by mutations in the F8 and F9 genes, resulting in quantitative or qualitative defects in factor VIII (FVIII) and factor IX (FIX), respectively. The prevalence of the disorders is 1 in 5000 (HA) and 1 in 30,000 (HB) male births . A recurring inversion located at intron 22 in F8 accounts for approximately 45% of all severe HA cases. Additionally, more than 2000 point mutations and indels distributed throughout the gene have been associated with the majority of the remaining HA cases (http://www.factorviii-db.org). More than 1000 point mutations and indels have been reported in F9 in patients with HB (http://www.factorix.org).