Application of a molecular diagnostic algorithm for haemophilia A and B using next-generation sequencing of entire F8, F9 and VWF genes

Abstract

Currently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.

Two-incision laparoscopic appendectomy for a severe hemophilia A child patient with coagulation factor VII deficiency

“…Laboratory examination of hemophilia patients usually revealed a prolonged APTT; however, a defect in the binding of factor VIII and von Willebrand factor should be excluded out before a clear diagnosis of hemophilia A. [ 12 – 14 ] In this boy, the laboratory examination revealed an APTT of 95.6 seconds, nearly 2 times of the normal value, which is in conformity with his history. A detailed examination was made to confirm the diagnosis of hemophilia for this boy.…”

Targeted re-sequencing of F8, F9 and VWF: Characterization of Ion Torrent data and clinical implications for mutation screening

“…However, it is an obvious shortcoming of the technique for which it will need to be corrected. In contrast, the Illumina platform was used by Bastida et al [8] to perform hybridization-based targeted resequencing of F8 , F9 and VWF . They determined their coverage over the same target regions to 90.5% with a read depth of 50X, without giving further details of the system characteristics.…”

“…The studies use different enrichment strategies of the target regions. AmpliSeq uses PCR amplification based on pools of primers whereas the Illumina system of Bastida et al [8] used hybridization-based enrichment. With the AmpliSeq strategy a higher coverage was achieved (99%), though at a cost of lower uniformity, necessitating a higher average read depth to reliably call variants across individuals [21].…”

“…As Sanger-based resequencing only sequences the phenotypically indicated gene, it can lead to incorrect diagnosing as well as therapy. Bastida et al [8], described the importance of differentiation between the VWD-2N and the mild/moderate HA phenotypes. They also demonstrated the value of using gene panels as no mutation was found in F8 in eight previously diagnosed mild HA cases.…”

“…Recently a number of next generation sequencing (NGS)-based studies have been used to re-sequence panels of genes associated with inherited bleeding disorders in a single workflow. Bastida et al [8] designed a gene panel targeting F8 , F9 and VWF on an Illumina MiSeq platform whereas Bastida et al [9] used a 23-gene panel to target both common and rare bleeding disorders using the same platform. The ThromboGenomics initiative targeted 63 genes associated with inherited bleeding, thrombotic and platelet disorders [10].…”