Which genetic mutation panels should I order to evaluate for hereditary (familial) hemophagocytic lymphohistiocytosis (HLH)?

Genetic Testing for Hemophagocytic Lymphohistiocytosis (HLH)

Order a comprehensive next-generation sequencing (NGS) panel that includes PRF1, UNC13D, STX11, STXBP2, and SH2D1A as the core genes, supplemented by functional testing (NK cell cytotoxicity and CD107a degranulation assay) to confirm pathogenicity of identified variants. 1

Primary Gene Panel to Order

Your genetic testing should target these specific genes in order of frequency:

• PRF1 (Familial HLH type 2) - most common, accounts for approximately 20-50% of familial cases 1, 2
• UNC13D (Familial HLH type 3) - second most common 1, 2
• STX11 (Familial HLH type 4) 1, 3
• STXBP2 (Familial HLH type 5) 1, 2
• SH2D1A (X-linked lymphoproliferative syndrome type 1) - critical in male patients with EBV-associated HLH 1

Together, FHL2 and FHL3 account for approximately 84% of familial HLH cases 2

Extended Gene Panel for Specific Clinical Scenarios

For male patients with EBV-driven HLH or lymphoproliferation, add these genes as they indicate X-linked lymphoproliferative syndromes:

• XIAP/BIRC4 (X-linked lymphoproliferative syndrome type 2) 1
• ITK (inducible T-cell kinase deficiency) 1
• CD27 (CD27 deficiency) 1
• MAGT1 (magnesium transporter 1 deficiency) 1

For patients with partial albinism or pigmentary abnormalities, include:

• RAB27A (Griscelli syndrome type 2) 1, 3
• LYST (Chediak-Higashi syndrome) 1, 4
• AP3B1 (Hermansky-Pudlak syndrome type 2) 1, 4

For patients with recurrent autoinflammation or enterocolitis, consider:

• NLRC4 (inflammasome-related HLH) 1

Essential Functional Testing to Order Simultaneously

Do not rely on genetic testing alone. Order these functional assays concurrently because they provide rapid diagnostic information while awaiting sequencing results and help validate variants of uncertain significance 1, 2:

• NK cell cytotoxicity assay - detects functional defects in cytotoxic granule release 1
• CD107a degranulation assay by flow cytometry - measures lysosomal degranulation capacity 1, 2
• Perforin expression by flow cytometry - specifically identifies FHL2 (PRF1 mutations) 1, 2
• SAP protein expression - identifies XLP1 (SH2D1A mutations) in male patients 1

Flow cytometry-based assays are highly specific and sensitive for diagnosis and can functionally validate novel variants identified by sequencing 2

Critical Timing Considerations

Never delay treatment while awaiting genetic test results. 1, 5 The decision to initiate HLH-directed therapy must be based on clinical and laboratory criteria, not genetic confirmation, as mortality ranges from 20-88% without prompt treatment 5

Genetic testing serves three purposes, none of which should delay treatment:

1. Confirming familial HLH to guide transplant decisions 1
2. Providing family counseling and screening 1
3. Identifying specific syndromes requiring tailored management 1

Who Requires Genetic Testing

Order genetic testing in these specific scenarios 1:

• Family history of consanguinity and/or HLH - highest yield
• Recurrent or relapsing HLH episodes - suggests hereditary form
• Young male adults with EBV infection or lymphoproliferation - screen for XLP
• Patients with partial albinism - suggests Griscelli or Chediak-Higashi syndrome
• HLH with unknown trigger - consider underlying genetic predisposition
• Age <18 years - higher likelihood of familial forms

Common Pitfalls to Avoid

The A91V mutation in PRF1 is a hypomorphic variant present in 4-17% of healthy Caucasians and should not be considered diagnostic of FHL2 in isolation 1. This variant may constitute a risk factor but requires additional pathogenic mutations or functional defects to confirm disease causation.

Approximately 40-70% of adult HLH cases are malignancy-associated, particularly lymphoma 1, 5. Do not assume a genetic cause without thoroughly excluding occult malignancy through PET-guided imaging and repetitive tissue sampling 1, 5

Functional assays may be unreliable if the patient has low NK cell counts or is receiving immunosuppressive therapy 6. In these cases, genetic testing becomes more critical, but interpretation requires caution.

Interpreting Results

Biallelic mutations (homozygous or compound heterozygous) in PRF1, UNC13D, STX11, or STXBP2 confirm familial HLH 1, 2. Monoallelic (heterozygous) mutations alone are insufficient for diagnosis unless functional testing confirms defective cytotoxicity 1

Hemizygous mutations in SH2D1A or XIAP in males confirm X-linked lymphoproliferative syndrome 1

Novel variants of uncertain significance require functional validation using assays that assess the effect of mutations on cellular function 6, 2. Flow cytometry-based degranulation assays help validate pathogenicity 2

Genetic Heterogeneity Considerations

Extensive genetic and allelic heterogeneity exists in FHL, with 76 different disease-causing mutations identified across the four main genes, including 51% novel mutations in some cohorts 2, 3. This underscores the importance of comprehensive NGS panels rather than single-gene testing.

Exome sequencing can simultaneously screen multiple HLH-associated genes and is particularly useful when targeted panels are negative 4