What genetic test is recommended for a child with severe hemophilia A?

Genetic Testing for Severe Hemophilia A in Children

For a child with severe hemophilia A, perform F8 gene sequencing that includes testing for the intron 22 inversion first (present in ~30% of severe cases), followed by complete sequencing of all exons, splice junctions, and the promoter region if the inversion is negative, with multiplex ligation-dependent probe amplification (MLPA) to detect large deletions. 1, 2

Recommended Testing Algorithm

First-Line Testing: Intron 22 Inversion

• Begin with intron 22 inversion testing using single-tube PCR, as this accounts for approximately 30% of severe hemophilia A cases and can be completed rapidly 1, 2
• The intron 1 inversion should also be assessed, though it is much less common (approximately 1% of cases) 2
• This initial screening can identify the causative mutation within 14-42 hours depending on laboratory capabilities 1

Second-Line Testing: Complete Gene Sequencing

If inversion testing is negative, proceed with:

• Systematic sequencing of all 26 exons of the F8 gene 1
• Sequencing of all splice junctions to detect donor/acceptor site mutations 3
• Promoter region sequencing 1
• This comprehensive approach uses 23 PCR products under standardized conditions and identifies mutations in approximately 98% of severe hemophilia A patients 1, 2

Third-Line Testing: Deletion Analysis

• Multiplex ligation-dependent probe amplification (MLPA) to detect large deletions that may be missed by standard sequencing 2
• Large deletions carry the highest inhibitor risk (100% in one cohort), making their identification clinically critical for risk stratification 2

Clinical Importance of Mutation Identification

Inhibitor Risk Stratification

The specific mutation type directly impacts inhibitor development risk, which is crucial for treatment planning:

• Large deletions detected by MLPA: 100% inhibitor risk 2
• Mutations in the A3 domain: 60% inhibitor risk 2
• Mutations in the C2 domain: 50% inhibitor risk 2
• Overall inhibitor prevalence in severe hemophilia A: 26% 2

Genetic Counseling and Family Studies

• Once the causative mutation is identified, accurate carrier testing becomes straightforward for female family members 1, 4
• Direct mutation detection is superior to indirect RFLP-based linkage analysis, which only identifies carriers in 58-94% of cases depending on polymorphism informativeness 4
• Rare cases of de novo mutations in both parents can occur, requiring careful interpretation 5

Common Pitfalls to Avoid

• Do not rely solely on standard sequencing without MLPA, as large deletions will be missed and these carry the highest inhibitor risk 2
• Do not skip intron 22 inversion testing in severe cases, as this rapid test identifies nearly one-third of patients and saves time and resources 1, 2
• Do not assume both parents are carriers in sporadic cases, as de novo mutations occur and can affect genetic counseling 5

Mutation Spectrum Findings

Studies have identified diverse mutation types in severe hemophilia A:

• Intron 22 inversions: ~30% 2
• Point mutations: Including missense, nonsense, and splice site mutations 3
• Large deletions: Detected by MLPA 2
• Novel mutations: Continuously identified, with 24 novel mutations found in one cohort of 100 patients 2