A sequential variant prioritization to identify genetic causes of hereditary angioedema in affected families from Spain and Portugal

Technical Summary: Sequential Variant Prioritization for Hereditary Angioedema in Spain and Portugal

Problem Statement
Hereditary angioedema (HAE) is a rare autosomal dominant disorder characterized by recurrent edema attacks mediated by bradykinin dysregulation. While the majority of cases are attributed to variants in SERPING1 (encoding C1 inhibitor) or F12 (encoding Factor XII), a significant proportion of patients—particularly those with normal C1 inhibitor levels (HAE-nC1INH)—lack a molecular diagnosis. Current diagnostic routines often rely on protein-level assays, which can be inconclusive or lead to misdiagnosis, resulting in delayed treatment. Furthermore, while Next-Generation Sequencing (NGS) has expanded the known genetic landscape, the diagnostic yield remains suboptimal, especially in sporadic cases without family history. There is a critical need for comprehensive genetic characterization to refine diagnostic strategies and expand the mutational spectrum of HAE in underrepresented populations, specifically within Spain and Portugal.

Methodology
The study employed a sequential variant prioritization strategy on a cohort of 102 independent families (170 individuals) from Spain and insular Portugal. The workflow involved:

1. Whole-Exome Sequencing (WES): DNA was extracted from peripheral blood and sequenced using Illumina instruments, achieving a median depth of 118.3X.
2. Two-Level Prioritization Pipeline:
   • Level 1 (Established Genes): Utilized the in-house Hereditary Angioedema Database Annotation (HADA) tool to screen seven known HAE-associated genes (SERPING1, F12, ANGPT1, PLG, KNG1, HS3ST6, MYOF). This level focused on identifying known pathogenic variants and novel variants within these established loci.
   • Level 2 (Exploratory Panel): For cases unresolved in Level 1, a virtual gene panel was applied using the Franklin platform. This panel targeted genes involved in the kinin-kallikrein system (KKS) and complement pathways (e.g., DAB2IP, C5, TEK, VEGFA), prioritizing variants with low population allele frequencies (<0.01) and high predicted deleteriousness (CADD scores exceeding gene-specific Mutational Significance Cutoffs).
3. Validation and Segregation: Candidate variants were validated via Sanger sequencing. Segregation analysis was performed where family members were available to assess co-segregation with the disease phenotype.
4. Specialized Analysis: The pipeline included specific screening for mobile element insertions (Alu elements) in SERPING1 using MELT and SCRAMble, and splicing impact prediction using SQUIRLS and DECIPHER.

Key Results

• Diagnostic Yield: The study identified 28 pathogenic or likely pathogenic (P/LP) variants. The diagnostic yield was significantly higher in multiplex families (88.3%) compared to proband-only cases (21.1%), underscoring the value of including unaffected family members in screening.
• Novel Discoveries: The analysis revealed seven novel SERPING1 variants and two novel F12 variants. Notable findings included a novel splicing variant in SERPING1 and a duplication event predicted to trigger Nonsense-Mediated Decay (NMD).
• Candidate Variants: In cases lacking mutations in established genes, the Level 2 analysis prioritized candidate variants in genes such as C5, TEK, ANGPT2, and KRT1. Specifically, a splice-site variant in C5 (c.3151_3154+2del) was found in four unrelated sporadic cases, and a TEK missense variant showed complete co-segregation in one family, suggesting a role in HAE-nC1INH pathogenesis.
• Epidemiological Observations: The study noted a striking prevalence of HAE type 2 (dysfunctional C1-INH) in Portuguese patients (40%), significantly higher than global estimates (10–15%).
• Reclassification: Genetic analysis successfully reclassified cases with ambiguous serological profiles, such as Family 97, where an initial clinical suspicion of HAE-C1INH type 1 was revised to HAE-nC1INH based on the identification of an F12 variant.

Significance and Claims
The authors claim that this study represents the most comprehensive genetic characterization of HAE in Spain and Portugal to date. By integrating WES with a sequential prioritization framework, the study expands the known mutational spectrum of HAE and proposes novel candidate variants in genes interacting with the bradykinin pathway. The paper asserts that genetic testing should be established as a first-level diagnostic tool to reduce diagnostic delays and enable precise management, particularly for patients with normal C1 inhibitor levels. The findings reinforce the importance of familial segregation analysis to improve diagnostic yield and highlight the limitations of current protein-based assays in resolving ambiguous cases. The authors emphasize that while the identified candidate variants in the second level require functional validation, they provide a framework for re-evaluating the molecular diversity of HAE and suggest that expanding genetic screening beyond canonical genes is necessary to address the remaining diagnostic gap.