C1-inhibitor gene nucleotide insertion causes type II hereditary angio-oedema

The polymerase chain reaction and nucleotide sequence analysis have been used to characterise a three nucleotide insertion in the eighth exon of one allele of the C1-inhibitor gene between nucleotides 16749 and 16750 in a kindred with type II hereditary angio-oedema (HAE). The effect of the resulting C1-inhibitor amino acid sequence alteration is discussed. This represents the first report of a nucleotide insertion in the C1-inhibitor gene causing type II HAE.     

Type II hereditary angio-oedema associated with two mutations in one allele of the C1-inhibitor gene around the reactive-site coding region.

The polymerase chain reaction and nucleotide sequence analysis have been used to characterise two point mutations in the eighth exon of one allele of the C1-inhibitor gene in a kindred with type II hereditary angio-oedema (HAE). The mutations comprise a G to A substitution at C1-inhibitor gene nucleotide 16789 and an upstream C to T substitution at nucleotide position 16765. This represents the first report of these two mutations in the same C1-inhibitor allele in type II HAE. The molecular genetic pathogenesis of HAE is discussed in the light of these findings.     

A point mutation in exon 7 of the C1-inhibitor gene causing type I hereditary angioedema

The polymerase chain reaction and nucleotide sequence analysis have been used to characterize a point mutation in the seventh exon of one allele of the C1-inhibitor gene in a family with type I hereditary angioedema. A single base change (C→T) at nucleotide 1482 in C1-inhibitor converted the codon for Gln-339 to a premature translation termination codon, TAG. Family studies suggest that this mutation is reponsible for type I hereditary angioedema in a studied pedigree. Received: 19 March 1996     

Presence of C1-Inhibitor Polymers in a Subset of Patients Suffering from Hereditary Angioedema

Hereditary angioedema (HAE) is a potentially life-threatening disease caused by mutations in the gene encoding the serine protease inhibitor (serpin) C1 inhibitor (C1-inh). The mutations cause decreased functional plasma levels of C1-inh, which triggers unpredictable recurrent edema attacks. Subjects suffering from HAE have been classified in type I patients with decreased functional and antigenic levels of C1-inh, and type II patients with decreased functional but normal antigenic C1-inh levels. However, a few reports have demonstrated that some mutations cause C1-inh polymerization in vitro, and it is speculated that C1-inh polymers may exist in patient plasma, challenging the current classification of HAE patients. To investigate the presence of C1-inh polymers in patient plasma samples, we developed an immunological method, where monoclonal antibodies produced against polymerized C1-inh were applied in native PAGE western blotting. Using this approach we analyzed genuine plasma samples from 31 Danish HAE families, and found that plasma samples from three genotypically distinct HAE type I families (classified upon C1-inh plasma concentrations) contained C1-inh polymers. Identical C1-inh polymerization phenotypes were observed in four affected family members from one of these families. Genotyping of the families revealed that the polymerogenic mutations of two families were located in proximity to the reactive center loop insertion site in C1-inh (p.Ile271Thr and p.Ser258_Pro260del),and one mutation affected helix C (p.Thr167Asn). In conclusion, we demonstrate that C1-inh polymers are present in the plasma of a subgroup of HAE type I patients.     

Rapid and sensitive techniques for identification and analysis of ''reactive-centre'' mutants of C1-inhibitor proteins contained in type II hereditary angio-oedema plasmas.

Novel procedures for structural analysis of the 'reactive-centre' residues, particularly the P1 residue, of the dysfunctional C1-inhibitor proteins found in the plasmas of type II hereditary angio-oedema (HAE) patients are described. C1-inhibitor is adsorbed directly from plasma on to Sepharose-anti-(C1 inhibitor) beads. The P1 residue of C1 inhibitor is arginine and hence a potential cleavage site for trypsin. Thus trypsin digestion of the immobilized protein, followed by SDS/PAGE of the released fragments, identifies P1 residue mutations. Pseudomonas aeruginosa elastase digestion of the immobilized protein, followed by purification of the released C-terminal peptide (by h.p.l.c.) and N-terminal sequence analysis defines the new P1 residue (or other mutations in the reactive-centre region). The techniques are both rapid and highly sensitive, requiring only 400 microliters of plasma. In addition, they permit accurate assessment of the level of normal (functional) inhibitor in a subclass of type II HAE plasmas, those containing P1-residue mutant proteins.     

Anti-cholesterol antibody levels in hereditary angioedema

Hereditary angioedema (HAE) is a rare disorder caused by the deficiency of the C1-inhibitor gene (C1INH) and characterized by recurrent bouts of angioedema. Autoimmune disorders frequently occur in HAE. Previously we found, that danazol has an adverse effect on serum lipid profile: reduced high-density lipoprotein (HDL) and elevated low-density lipoprotein (LDL) cholesterol levels are associated with long-term prophylactic use, whereas total cholesterol levels are unchanged. Our aim was to study the anti-cholesterol antibody (ACHA) production in HAE patients and compare it with those of healthy blood donors, and to investigate the possible associations between ACHA levels and serum lipid profile alterations caused by danazol. Anti-cholesterol IgG levels were measured by ELISA and their correlation with serum concentrations of total cholesterol, HDL, LDL, triglycerides was determined in HAE patients receiving/not receiving danazol. Serum ACHA levels were significantly higher in HAE patients, compared to healthy blood donors (P<0.0001). Longterm danazol prophylaxis had no effect on serum ACHA levels in HAE patients. However, we found a significant, negative correlation between ACHA levels and serum total cholesterol (r=-0.4033, P=0.0200), LDL (r=-0.4565, P=0.0076) and triglyceride (r=-0.4230, P=0.0121) levels only in danazol-treated patients, but not in HAE patients who did not receive long-term prophylaxis. Patients with HAE have higher baseline ACHA levels compared to healthy subjects, and this might reflect polyclonal B-cell activation. The latter would be a potential explanation for the lack of an increased incidence of infectious diseases in HAE patients, but might lead to increased autoimmunity.     

Exonic point mutations in NADH-cytochrome B5 reductase genes of homozygotes for hereditary methemoglobinemia, types I and III: Putative mechanisms of tissue-dependent enzyme deficiency

We analyzed the NADH-cytochrome b5 reductase gene of hereditary methemoglobinemia type I and type III, by using PCR-related techniques. The mutation in type I is a guanine-to-adenine substitution in codon 57 of exon 3 of the NADH-cytochrome b5 reductase gene, and the sense of this codon is changed from arginine to glutamine. In type III the mutation is a thymine-to-cytosine transition in codon 148 of exon 5, causing leucine-to-proline replacement in type III. The former mutation abolishes the MspI recognition site. Homozygosity for the former mutation in a patient with type I was confirmed by restriction analysis of PCR-amplified fragments and by dot blot hybridization of amplified products with allele-specific oligonucleotide probes. The latter mutation generates a recognition site for MspI. Amplification of exon 5 by PCR followed by digestion with MspI revealed homozygosity for this mutation in patients with type-III. Putative mechanisms of tissue-dependent enzyme defects in hereditary methemoglobinemia are discussed.     

A mutation in the pro alpha 2(I) gene (COL1A2) for type I procollagen in Ehlers-Danlos syndrome type VII: evidence suggesting that skipping of exon 6 in RNA splicing may be a common cause of the phenotype.

Fibroblasts from a proband with Ehlers-Danlos syndrome type VII synthesized approximately equal amounts of normal and shortened pro alpha 2(I) chains of type I procollagen. Nuclease S1 probe protection experiments with mRNA demonstrated that the pro alpha 2(I) chains were shortened because of a deletion of most or all of the 54 nucleotides in exon 6, the exon that contains codons for the cleavage site for procollagen N-proteinase. Sequencing of genomic clones revealed a single-base mutation that converted the first nucleotide of intron 6 from G to A. Therefore, the mutation was a change, in the -GT-consensus splice site, that produced efficient exon skipping. Allele-specific oligonucleotide hybridizations demonstrated that the proband's mother, father, and brother did not have the mutation. Therefore, the mutation was a sporadic one. Analysis of potential 5' splice sites in the 5' end of intron 6 indicated that none had favorable values by the two commonly employed techniques for evaluating such sites. The proband is the fourth reported proband with Ehlers-Danlos syndrome VII with a single-base mutation that causes skipping of exon 6 in the splicing of RNA from either the COL1A1 gene or COL1A2 gene. No other mutations in the two type I procollagen genes have been found in the syndrome. Therefore, such mutations may be a common cause of the phenotype. The primers developed should be useful in screening for the same or similar mutations causing the disease.     

Mutational spectrum of the C1INH (SERPING1) gene in patients with hereditary angioedema

Hereditary angioedema (HAE) is an autosomal dominant disease that manifests as intermittent acute swellings of the skin and mucosal surfaces, which, in the gastrointestinal tract and larynx, may even be fatal. HAE results from functional deficiency of the C1 inhibitor (C1INH) protein, which plays a key role in the classical pathway of complement activation. C1INH is the sole inhibitor of the activated proteases C1r and C1s, and is the major regulator of activated coagulation Factor XII and plasma kallikrein, which limits the generation of the vasoactive peptide bradykinin. In this paper, we report on the genetic analysis of 173 families (including 326 members) with a clinical diagnosis of HAE. Direct sequencing, Southern blotting and quantitative PCR by the MLPA method were used to screen for mutations in C1INH (SERPING1). In 142 families (82.1%), a causative C1INH gene mutation could be identified. A total of 80 novel point mutations of C1INH not published previously were detected in 96 pedigrees (including 172 members). Our results corroborate C1INH (SERPING1) deficiency as a disease of extreme allelic heterogeneity with almost each individual family carrying their own mutation. Routine molecular genetic analysis is an effective way of confirming the clinical diagnosis and identifying mutation carriers early on before any clinical manifestation becomes apparent. It is, therefore, a valuable tool in prevention and adequate treatment of acute and life-threatening oedema.     

An RNA splice site mutation in the C1-inhibitor gene causes type I hereditary angio-oedema

Summary Restriction fragment length polymorphism analysis, the polymerase chain reaction and nucleotide sequencing have been used to characterise a single base substitution (GT) at nucleotide 8863 in the C1-inhibitor gene. This destroys the 5 donor splice site recognition motif of the sixth intron. Family studies suggest that the mutation is responsible for type I hereditary angio-oedema in a studied kindred.     

Dysfunctional C1-inhibitor(At), isolated from a type II hereditary-angio-oedema plasma, contains a P1 ''reactive centre'' (Arg444----His) mutation.

Simple rapid procedures for identification and analysis of dysfunctional C1-inhibitor proteins mutated at the reactive-centre P1 residue have been developed and used to define structurally a C1-inhibitor protein, C1-inhibitor(At), isolated from an individual with hereditary angio-oedema. The observed mutation, Arg444----His, is compatible with a single base change in the codon used for Arg444 in the native protein.     

Recombinational biases in the rearranged C1-inhibitor genes of hereditary angioedema patients

DNA structural changes responsible for hereditary angioedema were sought in the C1-inhibitor gene, which contains unusually dense clusters of Alu repeats in various orientations. Among patients belonging to 45 unrelated families, eight partial C1-inhibitor gene deletions and a partial duplication were found. Four deletions had one of the boundaries within the gene and the other in extragenic regions--in three cases 5' of the gene and in one case 3' of the gene. The boundaries of the partial duplication and of the remaining four deletions mapped instead within a few kilobases of exon 4. The same element--Alu 1--the first of three tandem Alu repeats preceding exon 4, contained one of the breakpoints of each of these five rearrangements. Moreover, these recombination breakpoints spread over the entire length of Alu 1, in contrast with the tight clustering observed near the 5' end of Alu sequences rearranged in other human genes. Thus, two uncommon recombinational biases are observed in the Alu rearrangements of hereditary angioedema patients; one promotes the occurrence of intragenic breakpoints in a single Alu repeat, and the other allows the breaks to be distributed over the entire Alu structure rather than within the hot spot of the left Alu monomer. A region of potential Z-DNA structure, located 1.7 kb upstream of Alu 1, may contribute to both peculiarities.     

Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody

Plasma kallikrein (pKal) proteolytically cleaves high molecular weight kininogen to generate the potent vasodilator and the pro-inflammatory peptide, bradykinin. pKal activity is tightly regulated in healthy individuals by the serpin C1-inhibitor, but individuals with hereditary angioedema (HAE) are deficient in C1-inhibitor and consequently exhibit excessive bradykinin generation that in turn causes debilitating and potentially fatal swelling attacks. To develop a potential therapeutic agent for HAE and other pKal-mediated disorders, we used phage display to discover a fully human IgG1 monoclonal antibody (DX-2930) against pKal. In vitro experiments demonstrated that DX-2930 potently inhibits active pKal (K_i = 0.120 ± 0.005 nm) but does not target either the zymogen (prekallikrein) or any other serine protease tested. These findings are supported by a 2.1-Å resolution crystal structure of pKal complexed to a DX-2930 Fab construct, which establishes that the pKal active site is fully occluded by the antibody. DX-2930 injected subcutaneously into cynomolgus monkeys exhibited a long half-life (t_½ ∼12.5 days) and blocked high molecular weight kininogen proteolysis in activated plasma in a dose- and time-dependent manner. Furthermore, subcutaneous DX-2930 reduced carrageenan-induced paw edema in rats. A potent and long acting inhibitor of pKal activity could be an effective treatment option for pKal-mediated diseases, such as HAE.     

Analysis of the SMN and NAIP genes in slovak spinal muscular atrophy patients

We identified homozygous absence of exon 7 of the telomeric copy of the survival motor neuron gene (telSMN) in 88.4% (38/43) of spinal muscular atrophy (SMA) patients from Slovakia. Additional deletions within the neuronal apoptosis inhibitory protein (NAIP) gene were found in 38.5% of type I, 12.5% of type II and never in type III SMA patients. Neither the SMN nor the NAIP gene was deleted in 81 healthy relatives and 25 controls tested. In one family, pseudodominant inheritance was identified. Both the type III SMA father and type II SMA son carried the homozygous deletion of the telSMN gene. One SMA I patient showed an SMN hybrid gene, probably created by intrachromosomal deletion. In two haploidentical type II SMA sibs, the telSMN exon 7 was absent on one chromosome, while the other carried an A-->G transition 96 bp upstream of exon 7 of the telSMN gene, a potential disease-causing mutation in these patients.     

Acceptor splice site mutation in the invariant AG of intron 5 of the protein C gene,causing type I protein C deficiency

An acceptor splice-site mutation (3318, AG) in the invariant AG of intron 5 of the human protein C gene has been identified in a Spanish family with heterozygous type I protein C (PC) deficiency and thromboembolic disease. Family studies confirmed cosegregation of the mutation with type I PC deficiency. Computer analysis of the mutated sequence predicted the normal splicing site to be abolished by this mutation, whereas a cryptic splice site located two nucleotides downstream, in exon 6, is probably activated. According to this, 3318, AG should result in a frameshift with a stop at codon 119, in agreement with the presence of a type I or quantitative PC deficient phenotype in the affected members of the family.     

Osteogenesis imperfecta type I: molecular heterogeneity for COL1A1 null alleles of type I collagen.

Osteogenesis imperfecta (OI) type I is the mildest form of inherited brittle-bone disease. Dermal fibroblasts from most affected individuals produce about half the usual amount of type I procollagen, as a result of a COL1A1 "null" allele. Using PCR amplification of genomic DNA from affected individuals, followed by denaturing gradient gel electrophoresis (DGGE) and SSCP, we identified seven different COL1A1 gene mutations in eight unrelated families with OI type I. Three families have single nucleotide substitutions that alter 5' donor splice sites; two of these unrelated families have the same mutation. One family has a point mutation, in an exon, that creates a premature termination codon, and four have small deletions or insertions, within exons, that create translational frameshifts and new termination codons downstream of the mutation sites. Each mutation leads to both marked reduction in steady-state levels of mRNA from the mutant allele and a quantitative decrease in type I procollagen production. Our data demonstrate that different molecular mechanisms that have the same effect on type I collagen production result in the same clinical phenotype.