To the editor,
Hereditary angioedema (HAE), an autosomal dominant disease, shows considerable heterogeneity of manifestations, ranging from being asymptomatic or frequent episodes of edema of the extremities to life-threatening upper airway edema. In China, 60.8% of HAE patients experience airway mucosal swelling; of these patients, 9% die from it.1 Unfortunately, there are no effective biomarkers or modifiers that can predict the severity of HAE. Several studies have focused on the relationship between the genotype and the phenotype in HAE.2,3,4,5 We published nonsense and frameshift mutation, and mutations in Arg466 that affected the antigenic level of C1 esterase inhibitor (C1-INH) but were not associated with the severity of HAE.3 A previous study reported that c.-21T>C could impact C1-INH mRNA levels and might influence disease severity,2 but another study demonstrated that c.-21T>C was not associated with a more severe manifestation.5 Our previous work on the C1-INH gene in a single HAE family showed that some subjects carried not only the hereditary mutation but also an extra mutation or single nucleotide polymorphism. Whether these compound mutations aggravate C1-INH deficiency and the severity of HAE is unknown. Therefore, we selected 2 types of compound mutations and compared their impact on C1-INH deficiency in vitro. The methods were described in Supplementary Material.
Four patients with type I HAE originated from 2 families. Their clinical information is shown in Table. Four genotypes were identified (Table); c.1328A<G+c.167T<C (A1), c.1328A<G (A2), c.953C<G+c.49G<A (B1), and c.953C<G (B2). Compared with healthy control subjects, the C1-INH mRNA of patients A1, A2, B1, and B2 was 0.5%, 0.7%, 0.5%, and 8.1% of normal levels, respectively. By ligating C1-INH cDNA to M2 plasmid, transfecting them into Trans1-T1 cells, and picking single clone for sequencing, we detected both the compound mutations were located in the same mRNA transcript, namely, in cis to each other. Recombinated plasmid carrying 4 C1-INH genotypes were expressed in the Hep G2 cell line. Functional analysis showed that all genotypes expressed lower functional C1-INH compared with the normal control. The functional levels of C1-INH in genotypes in A1, A2, B1, and B2 were 0.34±0.13, 0.24±0.02, 0.44±0.06, and 0.11±0.03 U C1-INH/mL, respectively. By statistical analysis, c.49G<A+c.953C<G resulted in significantly lower function of recombinant C1-INH than c.953C<G (P<0.05). However, this difference was not observed between c.167T<C+c.1328A<G and c.1328A<G. Thus, it is inferred that c.49G<A might aggravate the dysfunctional level of C1-INH caused by c.953C<G. In terms of clinical symptoms, patient B1 expressed a severe phenotype, experiencing recurrent episodes that involved the upper airway, extremities, and face, whereas patient B2 presented only with several attacks hands edema during pregnancy and face swelling after trauma. However, since only 1 case with additional c.49G<A had severe symptoms in this study, it was hard to draw a conclusion that c.49G<A is associated with severe HAE phenotype in c.953C<G carriers. More proof studies are needed to confirm our results.
Table. Clinical and genetic information of the patients.
| Patients' number | Gender | Age (yr) | C1-INHa (g/L) | C4 (g/L) | Manifestations | Mutations | Molecular consequence | mRNA level* |
|---|---|---|---|---|---|---|---|---|
| A1 | Male | 33 | 0.09 | 0.061 | UAE (2–3 episodes/year), extremities | c.1328A<G | p.H443R | 0.51 |
| c.167T<C | SNP | |||||||
| A2 | Female | 11 | 0.1 | 0.081 | Twice hands edema | c.1328A<G | p.H443R | 0.76 |
| B1 | Female | 41 | 0.06 | 0.05 | UAE (once per month), face | c.953C<G | p.S318× | 0.5 |
| c.49G<A | p.G17R | |||||||
| B2 | Female | 31 | 0.04 | 0.018 | Hands edema during pregnancy, face swelling after head trauma | c.953C<G | p.S318× | 8.13 |
C1-INHa, C1 esterase inhibitor antigen level; UAE, upper airway edema.
*C1-INH mRNA relative quantity by real-time polymerase chain reaction.
In summary, the mutant genotypes that we identified in this study impaired C1-INH function in Hep G2 cells, and the level of functional C1-INH with the compound mutation c.953C<G+ c.49G<A was significantly lower compared with c.953C<G alone.
ACKNOWLEDGMENTS
This research was supported by National Natural Science Foundation of China (No. 81472870), CAMS Innovation Fund for Medical Sciences (CIFMS, No. 2016-I2M-1-002), and The National Key Research and Development Program of China (No. 2016YFC0901501).
Footnotes
There are no financial or other issues that might lead to conflict of interest.
SUPPLEMENTARY MATERIALS
Pedigrees in two HAE families.
Functional levels of recombinated C1 inhibitor (C1-INH). The median ± SD of functional levels for A1, A2, B1 and B2 were 0.34±0.13, 0.24±0.02, 0.44±0.06 and 0.11±0.03 U C1-INH/mL. A1, c.1328A<G+ c.167T<C; A2, c.1328A<G; B1, c.953C<G+c.49G<A; B2, c.953C<G.
References
- 1.Xu YY, Zhi YX, Liu RL, Craig T, Zhang HY. Upper airway edema in 43 patients with hereditary angioedema. Ann Allergy Asthma Immunol. 2014;112:539–544. doi: 10.1016/j.anai.2014.03.003. [DOI] [PubMed] [Google Scholar]
- 2.Duponchel C, Djenouhat K, Frémeaux-Bacchi V, Monnier N, Drouet C, Tosi M. Functional analysis of splicing mutations and of an exon 2 polymorphic variant of SERPING1/C1NH. Hum Mutat. 2006;27:295–296. doi: 10.1002/humu.9414. [DOI] [PubMed] [Google Scholar]
- 3.Xu YY, Zhi YX, Yin J, Wang LL, Wen LP, Gu JQ, et al. Mutational spectrum and geno-phenotype correlation in Chinese families with hereditary angioedema. Allergy. 2012;67:1430–1436. doi: 10.1111/all.12024. [DOI] [PubMed] [Google Scholar]
- 4.Bygum A, Fagerberg CR, Ponard D, Monnier N, Lunardi J, Drouet C. Mutational spectrum and phenotypes in Danish families with hereditary angioedema because of C1 inhibitor deficiency. Allergy. 2011;66:76–84. doi: 10.1111/j.1398-9995.2010.02456.x. [DOI] [PubMed] [Google Scholar]
- 5.Cumming SA, Halsall DJ, Ewan PW, Lomas DA. The effect of sequence variations within the coding region of the C1 inhibitor gene on disease expression and protein function in families with hereditary angioedema. J Med Genet. 2003;40:e114. doi: 10.1136/jmg.40.10.e114. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
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Supplementary Materials
Pedigrees in two HAE families.
Functional levels of recombinated C1 inhibitor (C1-INH). The median ± SD of functional levels for A1, A2, B1 and B2 were 0.34±0.13, 0.24±0.02, 0.44±0.06 and 0.11±0.03 U C1-INH/mL. A1, c.1328A<G+ c.167T<C; A2, c.1328A<G; B1, c.953C<G+c.49G<A; B2, c.953C<G.