Molecular pathogenesis and therapeutic advances in haemophilia, an update of the current evidence

Table 1 Genomic and structural characteristics of the F8 and F9 genes of their variant databases

Feature	F8 gene	F9 gene
Chromosomal location	Xq28	Xq27
Gene size	Approximately 186 kb[11]	Approximately 34 kb[11]
Number of exons	26	8
Protein encoded	Coagulation factor VIII	Coagulation factor IX
Protein structure/domains	A1-a1-A2-a2-B-a3-A3-C1-C2; encoded partly by large exon 14[11]	Signal peptide, propeptide, Gla domain, EGF1, EGF2, activation peptide, serine protease domain[11]
Key genomic features	Intron 22 region containing intronic gene copies (F8A and F8B), predisposing to Inv22 (40%-50% of severe HA)[11]; intron 1 low-copy repeats predisposing to Inv1 (2%-5% of severe HA); CpG hotspots accounting for approximately 40% of small variants despite comprising approximately 2% of sequence[11]	Compact gene with modular exon-domain correspondence; promoter contains androgen-responsive elements responsible for haemophilia B Leyden phenotype[11]
Predominant mutation classes	Inv22 (40%-50% of severe); Inv1 (2%-5% of severe); nonsense, frameshift, splice-site defects dominate in severe HA; missense variants common in mild/moderate HA	Missense (approximately 68%) dominate; nonsense/frameshift variants associated with severe phenotype; splice-site variants relatively frequent; large deletions rare (1%-3%) but high inhibitor risk
Mutation hotspots	Inv22 and Inv1 mediated by homologous recombination	Noncomparable to F8 inversions; mutations distributed across gene
Typical diagnostic methods	Long-range PCR for inversion detection; Sanger/NGS sequencing for point mutations, indels; MLPA for deletions/duplications	Direct sequencing (gene small enough for full coverage); MLPA when deletions suspected

HA: Haemophilia A; Inv22: Intron 22 inversion; Inv1: Intron 1 inversion; NGS: Next gene sequence; MLPA: Multiplex ligation-dependent probe amplification.

Table 2 Mutational spectrum of hemophilia A (F8 variants) and associated phenotypes

Mutation type	Approximate distribution	Associated severity	Molecular mechanism
Inv22[15-20]	40%-50% of severe HA globally; lower in some regions (e.g., 10.5% in Albania, approximately 30% in parts of India/Asia)	Severe	Homologous recombination between int22h-1 and extragenic int22h-2/int22h-3 repeats - disrupted F8 transcription
Inv1[15-17]	2%-5% of severe HA	Severe	Homologous recombination within intron 1
Nonsense mutations[14]	Common among severe, inversion-negative HA	Severe	Premature stop codons - truncated nonfunctional FVIII; often triggers NMD
Frameshift mutations (small insertions/deletions)[17-19]	Frequent in severe HA; multiple novel variants identified in several populations	Severe	Reading-frame disruption - premature truncation
Canonical splice-site mutations[14,17]	Common among severe phenotypes	Severe	Aberrant splicing - exon skipping or truncation
Missense mutations[16,17,22]	Predominant in mild and moderate A; smaller contribution to severe HA (especially at conserved residues)	Mild-moderate; occasionally severe	Residue substitution affects FVIII structure, stability, or cofactor function
Small deletions/insertions (non-frameshift)[22]	Less common but clinically significant	Mild-severe depending on domain affected	Disruption of local protein domains without full truncation
Large deletions/multiexon deletions[21]	Rare	Severe	Loss of entire domains - absent FVIII
Composite (double) mutations[18]	Rare	Severe	Combined effects of two pathogenic variants

HA: Haemophilia A; Inv22: Intron 22 inversion; Inv1: Intron 1 inversion; NMD: Nonsense mutational deletions.

Table 3 Mutational spectrum of haemophilia B (F9 variants) and structural consequences

Mutation type	Approximate distribution	Associated severity	Structural mechanism
Missense variants[11,23-25]	Approximately 68% of all F9 variants; the dominant mutation class[11]	Mild-moderate most commonly; sometimes severe when essential residues affected	Alter FIX folding, γ-carboxylation, calcium binding, catalytic triad stability, or activation peptide processing
Nonsense mutations[25,28]	Less common than missense; significant proportion in severe HB	Severe	Premature truncation - absent or unstable FIX protein; NMD frequently triggered
Frameshift variants (insertions/deletions)[25]	Relatively uncommon but clinically important	Severe	Frameshift - truncated nonfunctional protein
Canonical splice-site mutations[25,28]	Common among severe HB due to compact exon-domain architecture	Severe	Aberrant splicing - exon skipping, truncated proteins, or defective post-translational processing
Promoter mutations (including HB Leyden)[11]	Rare overall	Childhood severe - spontaneous improvement after puberty	Androgen-responsive elements regulate transcription; puberty FIX expression
Synonymous pathogenic variants[27]	Rare but increasingly recognized	Mild-severe depending on impact on mRNA	Alter mRNA structure, translation rate, and co-translational folding (e.g., p.Val107Val)
Large deletions (partial or whole-gene)[29]	1%-3% of F9 mutations	Severe	Loss of entire exons or full gene; often destabilizes neighboring genomic regions
Contiguous gene deletion syndromes[29]	Extremely rare	Severe haemophilia + syndromic features	Deletion of F9 plus adjacent genes - multi-system phenotype

HB: Haemophilia B; NMD: Nonsense mutational deletions.

Table 4 Comparison of approved gene therapies for haemophilia A and B

Feature	Valoctocogene roxaparvovec (roctavian)[46,50,52,53]	Etranacogene dezaparvovec (hemgenix)[47-50]
Indication	HA	HB
Vector capsid	AAV5	AAV5
Transgene	B-domain-deleted FVIII, codon-optimized FVIII-SQ	FIX-Padua variant (R338 L), approximately 5-10 × higher specific activity
Mechanism of action	Hepatic expression of FVIII-SQ leads to endogenous FVIII production sufficient to convert severe HA to mild/normal range	Hepatic expression of FIX-Padua generates supraphysiologic FIX activity at low vector doses
Peak factor levels	Median FVIII 11.9%-62.3% at weeks 49-52 in phase 3	Approximately 30 IU/mL at 12 months across trials
Long-term factor expression	Decline over time typical: Approximately 50%-60% reduction from peak by 24 months	FIX expression more stable than FVIII; long-term persistence observed
Reduction in ABR	Meta-analysis: -7.58 treated bleeds/year; > 90% reduction in factor use	Meta-analysis: 5.64-fold ABR reduction; near-universal cessation of prophylaxis
Proportion of patients stopping prophylaxis	Majority (> 90%) discontinue FVIII prophylaxis	Majority discontinue FIX prophylaxis; FIX activity sufficient for stable haemostasis
Durability and challenges	Expression decline over several years; FVIII synthesis is hepatocyte-stressful (UPR/ER stress)	Durable expression; lower dose requirement improves safety margin
Key safety issues	Transaminase elevations common; very high vector dose raises hepatotoxicity concerns potential genomic integration events	ALT elevations possible but generally manageable lower vector dose reduces risk of hepatotoxicity
Advantages	First approved gene therapy for HA; major reductions in bleeding and factor use; high initial FVIII expression	High efficiency due to Padua transgene; stable FIX expression; very low dosing requirements

HA: Haemophilia A; HB: Haemophilia B; ALT: Alanine aminotransferase; ABR: Annualized bleeding rate.