For decades, a 28-year-old woman suffered from recurrent, painful swelling episodes. Standard genetic testing had failed to provide answers—until scientists looked deeper.
Hereditary angioedema (HAE) is a rare and potentially life-threatening genetic disorder characterized by sudden, recurrent episodes of severe swelling in various body parts. For approximately 5% of patients with HAE due to C1-inhibitor deficiency, standard genetic testing reveals no abnormalities in the SERPING1 gene—the very gene known to cause their condition 1 . These patients faced a diagnostic odyssey with no clear end in sight, until researchers began exploring the hidden regions of our DNA and discovered deep intronic mutations—variations occurring far from the typical areas examined in conventional testing. This article explores how these elusive mutations, particularly within the SERPING1 gene, cause hereditary angioedema and how scientists are developing innovative methods to detect them.
Hereditary angioedema is an autosomal dominant genetic disorder that affects approximately 1 in 50,000 people worldwide, without known ethnic differences 3 7 . The disease is characterized by unpredictable bouts of swelling that typically affect the limbs, face, gastrointestinal tract, and airways. Laryngeal edema represents the most feared complication, as it can lead to fatal asphyxiation if not properly managed 4 .
The underlying mechanism involves excessive bradykinin production, a powerful vasodilator that increases vascular permeability, leading to fluid leakage into tissues and subsequent swelling 3 . This process occurs due to deficiencies in the regulation of the kallikrein-kinin system, which is normally kept in check by the C1 inhibitor protein.
The SERPING1 gene, located on chromosome 11, provides instructions for making the C1 inhibitor (C1-INH) protein 3 . This protein plays a crucial role in regulating multiple systems in the body, including:
To date, over 900 different variants of the SERPING1 gene have been identified in HAE patients worldwide 2 7 .
Features normal or elevated antigenic C1-INH levels but reduced function, usually caused by missense mutations affecting the protein's reactive center 2 .
Genes consist of both exons (protein-coding regions) and introns (non-coding intervening sequences). Conventional genetic testing typically focuses on exons and the immediate boundaries between exons and introns. Deep intronic mutations occur within introns, far from these traditional boundary regions, and are therefore missed by standard genetic analyses 1 .
For many years, the role of these deep intronic regions remained poorly understood. However, advanced sequencing technologies have revealed that variations in these areas can significantly impact gene function by:
The discovery of deep intronic mutations in SERPING1 has solved diagnostic mysteries for many HAE families. In a groundbreaking 2020 study, researchers identified the novel deep intronic mutation c.-22-155G>T in intron 1 of the SERPING1 gene in two unrelated patients who had previously tested negative for mutations in the coding region 1 .
Through rigorous analysis, they demonstrated that this mutation caused degradation of the mutated allele, resulting in haploinsufficiency—where a single functional copy of the gene cannot produce sufficient C1-INH protein to maintain normal physiological control 1 .
Similarly, another study revealed a different deep intronic mutation (c.1029+384A>G) in intron 6 that created a de novo donor splice site, leading to pseudoexon inclusion in the final mRNA transcript—the first such mechanism described in SERPING1 5 .
The proximal part of intron 6 appears particularly prone to pseudoexon-activating mutations, as natural alternative exons and additional cryptic sites occur in this region 5 .
When standard approaches failed to identify mutations in 14 unrelated C1-INH-HAE patients, researchers employed a sophisticated diagnostic pipeline to uncover the hidden genetic defects 1 :
Using a next-generation sequencing (NGS) platform targeting the entire SERPING1 gene, including non-coding regions that conventional genotyping misses.
Detected variants with a global minor allele frequency lower than the frequency of C1-INH-HAE (0.002%) were selected for further analysis.
Ten different bioinformatics tools were used to predict the potential deleterious effects of the filtered variants.
Family studies were conducted to check for co-segregation of the variant with the disease across generations.
The pathogenic effect on RNA was examined by analyzing cDNA to demonstrate functional consequences of the mutation.
The investigation yielded crucial findings:
| Method | Detection Capability | Limitations |
|---|---|---|
| Sanger Sequencing | Effective for coding regions and exon-intron boundaries | Misses deep intronic variants, large deletions/duplications |
| Multiplex Ligation-dependent Probe Amplification (MLPA) | Detects large deletions/duplications of one or more exons | Cannot identify single nucleotide changes in introns |
| Next-Generation Sequencing (NGS) | Can sequence entire gene, including intronic regions | May require additional validation methods |
| RNA Analysis | Directly assesses functional impact on transcription | Requires appropriate tissue sample (e.g., blood) |
The discovery of deep intronic mutations in SERPING1 has transformed the diagnostic landscape for HAE:
A 2023 study of 207 Czech HAE patients from 85 families demonstrated the power of a systematic approach to SERPING1 defect screening 3 . Researchers employed:
This comprehensive strategy allowed them to detect a causal variant in all families except one, identifying 56 different variants, including 5 novel likely pathogenic variants 3 .
| Mutation Type | Percentage | Effect on Protein |
|---|---|---|
| Missense and in-frame | 36.8% | Amino acid changes or single codon adjustments |
| Frameshift | 28.9% | Premature stop codons and truncated proteins |
| Nonsense | 14.5% | Introduction of early stop codon |
| Splice site | 13.2% | Disrupted RNA splicing |
| Gross deletions/duplications | 6.6% | Partial or complete gene deletion |
Understanding deep intronic mutations requires specialized laboratory tools and techniques. The following table outlines essential resources used in this research area.
| Tool/Technique | Function | Application Example |
|---|---|---|
| Next-Generation Sequencing | High-throughput sequencing of entire genes/genomes | Identifying deep intronic variants in SERPING1 1 |
| Sanger Sequencing | Gold standard for validating DNA sequence variations | Confirming NGS findings and testing family members 4 |
| Multiplex Ligation-dependent Probe Amplification | Detecting exon-level copy number variations | Identifying large deletions/duplications in SERPING1 2 |
| RNA Analysis | Studying effects on mRNA splicing and stability | Demonstrating pseudoexon inclusion due to intronic mutations 5 |
| Minigene Assays | Functional analysis of splicing variants in vitro | Investigating impact of intronic variants on splicing mechanisms 3 |
| In Silico Prediction Tools | Computational prediction of variant pathogenicity | Initial assessment of newly discovered intronic variants 1 |
Advanced sequencing techniques reveal hidden mutations
Computational tools predict variant effects
Laboratory tests validate mutation impact
The discovery of deep intronic mutations in SERPING1 has revolutionized our understanding of hereditary angioedema genetics, providing answers to previously unsolved cases and highlighting the importance of looking beyond conventional coding regions.
As one research team concluded, "advanced DNA sequencing methods should be performed in cases of C1-INH-HAE where standard approaches fail to uncover the genetic alteration" 1 .
Genetic testing for HAE should increasingly include comprehensive sequencing of non-coding regions.
Understanding the specific molecular defect may guide targeted therapeutic approaches.
Accurate genetic diagnosis enables informed reproductive decisions for affected families.
As research continues, scientists are increasingly recognizing that much of the "dark matter" of the genome—including deep intronic regions—plays crucial roles in human health and disease. The story of SERPING1 and hereditary angioedema serves as a powerful example of how exploring these hidden genetic landscapes can illuminate diagnostic mysteries and offer new hope to patients and their families.