Reading between the (long) lines: one-stop F8 gene analysis? Open Access

In the context of strong genotype-phenotype correlations related to disease severity and inhibitor formation, as well as the advent of the new generation of treatments including gene therapy, achieving a prompt and precise molecular diagnosis of patients who harbor disease-causing variants in the F8 gene is essential.¹ Identification of these variants proves to be a challenge due to the wide variety of variant types reported, including inversions and other complex alterations involving large, highly homologous regions on the X chromosome. A large proportion of patients with severe hemophilia A harbor inversions involving introns 1 and 22, which cannot be identified using standard next-generation sequencing (NGS) assays, and a growing number of deep intronic variants are being described in patients with hemophilia A of all severities.² 

The current testing strategy for males with severe hemophilia A or females in whom the familial variant is not previously known, is complex and requires testing with multiple assay methodologies: one, to detect inversions and the other, to study single nucleotide variants (SNVs), deletion-insertions (delins), and copy number variants (CNVs). This approach is expensive and time consuming. Failing to test using different methods can provide false reassurance when a genetic variant is not identified. Moreover, the current standard assays miss the identification of the causative variant in up to 5% of people with hemophilia A (1%-2% of severe cases³ and 5%-6% of mild cases⁴), with complex structural variations and intronic variants unable to be detected.

In this issue of Blood Advances, Liu et al⁵ presents the use of a hybridization capture long-read sequencing (hc-LRS) platform for the detection of inversions, complex structural variation, SNVs, and delins using a single testing methodology. The authors were able to detect F8 intron 22–related inversions in 10 patients with severe hemophilia A, 2 of whom had complex rearrangements. The 2 patients with complex rearrangements may have been missed or inaccurately classified with conventional long-range polymerase chain reaction (LR-PCR) or inverse PCR methods. SNVs and CNVs were also adequately identified. Through pre-enrichment of the target regions, they are able to shorten the analytical time and reduce costs in the wet-laboratory portion of the assay. In addition, the hc-LRS provides greater depth of coverage than whole genome sequencing.

We are still, however, far from true comprehensive genetic analysis of F8. Despite the ability of hc-LRS to identify complex structural variation, additional methods such as LR-PCR and optical genome mapping were required to fully characterize complex structural variants. Additionally, this particular method likely misses potential duplications in high homology regions, as was noted in samples A10-2 and A12, in which subsequent confirmatory testing was performed. Although the cost-reduction potential is clear, multiplexing to achieve the quoted $100 per sample requires large testing volumes, which most clinical laboratories, even reference laboratories, will be unable to achieve. The need to batch samples would then negate the benefit of faster analytic times.

There is room for improvement by adding more probes to the regions surrounding the intron 22 homology regions (int22h-2 and int22h-3), aiming for longer read length and improving capture efficiency, which could overcome some of the challenges related to the characterization of complex structural variation in F8. In addition to technical optimization, careful consideration would need to be given to how this testing methodology is incorporated in the diagnostic approach of patients with bleeding and/or decreased factor VIII levels, considering the broad availability, familiarity, and versatility of NGS testing, particularly in areas of the world such as the Unites States where patients are evaluated in a variety of academic and community clinical settings and in others where cost continues to be a barrier.

Conflict-of-interest disclosure: The authors declare no competing financial interests.