Detection of C1 inhibitor mutations in patients with hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) results from a deficiency in the functional level of C1 inhibitor caused by mutations in the C1 inhibitor gene. The mutations responsible for HAE have been shown to be heterogeneous. OBJECTIVE: Because the identification of C1 inhibitor mutations may depend, in part, on the technique used to screen for mutations, we screened the entire C1 inhibitor coding region to identify mutations in a cohort of patients with HAE. METHODS: By using single-stranded conformational polymorphism analysis, 24 subjects with HAE from 16 different kindreds were screened for C1 inhibitor polymorphisms. C1 inhibitor mutations were identified by sequencing the exons containing identified polymorphisms. RESULTS: All 24 subjects with HAE had identifiable polymorphisms, involving exons 2, 3, 4, 5, or 8. Fourteen different C1 inhibitor mutations were identified: 8 missense, 1 nonsense, 4 frameshift, and 1 small deletion mutations. No large deletions or duplications were found. Nine of the 14 mutations represent newly recognized C1 inhibitor mutations, 6 of which involve exon 4. CONCLUSIONS: Single-stranded conformational polymorphism is an effective approach for identifying new mutations in HAE. Elucidation of the range of C1 inhibitor mutations causing HAE is important for both defining which residues are required for C1 inhibitor secretion or function and providing the basis for future studies to define the relationship between the C1 inhibitor genotype and disease severity.

Improved method for measuring C1-r-C1-s-(C1 inh)2 complexes by an enzyme-linked immunosorbent assay.

Measurement of C1-r-C1-s-(C1 inh)2 complexes in serum or plasma by enzyme-linked immunosorbent assay (ELISA) has been proposed as a relatively convenient and sensitive means for assessing C1 activation. However, interference by unactivated C1q (r-s)2 at low serum or plasma dilutions has resulted in estimates that vary widely with the degree of serum or plasma dilution. Precipitating the interfering C1q (r-s)2 with 6% polyethylene glycol has been proposed to resolve this problem, but here it is shown that this procedure also precipitates or coprecipitates some of the C1-r-C1-s-(C1 inh)2 complexes. Satisfactory results have been achieved without PEG precipitation by testing high plasma dilutions under conditions where there is a sufficient excess of anti-C1s coating the microtitration plate wells that removal of C1q (r-s)2 is not necessary. Optimizing conditions for quantitating these complexes at high dilution have been investigated. The mean normal EDTA plasma C1-r-C1-s-(C1 inh)2 complex measurement was 36.6 +/- 7.0 (S.D.) ELISA units with a 95% confidence interval of 19.5-47.6u. Besides providing a sensitive assay for C1 activation, measuring C1-r-C1-s-(C1 inh)2 complexes may help to clarify the pathophysiologic mechanisms resulting from C1 inh deficiency under various conditions.

Cold-dependent activation of complement: recognition, assessment, and mechanism.

Cold-dependent activation of complement (CDAC) is a phenomenon characterized by low hemolytic complement activity in chilled serum. Complement component levels are normal when measured immunologically, and there is normal hemolytic activity in EDTA plasma or serum maintained at 37 degrees C. Little attention has been paid to CDAC except in Japan, and current unfamiliarity with it, even by clinical immunologists, can lead to confusion and unnecessary laboratory tests. A 66-year-old patient with a complex medical history is described whose complement tests showed abnormalities characteristic of CDAC. Evidence for classical complement pathway activation in the cold was obtained by CH50 measurements, by hemolytic C4 determinations, by C4a, C3a, and C4d generation, and by quantitating C1s-C1r-(C1 inhibitor)2 complexes. A good correlation was observed among these parameters. Cryoprecipitates were absent. CDAC activity has persisted for over 5 years and is greater at 13 than at 4 degrees C. Activation is ablated by heating at 56 degrees C and restored by the addition of C1 to the heated serum. Adsorption by streptococcal protein G-Sepharose and precipitation by 2.5% polyethylene glycol support the hypothesis that CDAC is caused by aggregated IgG. The CDAC factor(s) also induces complement activation in normal serum but has not interfered with Raji cell or C1q binding tests or with FACS analysis. More limited studies of a second individual experiencing CDAC yielded similar results.