Following the publication of the original article [1], the authors have requested to amend the Abstract and Discussion section as follows.

Acquired generalized lipodystrophy (AGL) is a rare condition characterized by an altered distribution of adipose tissue and predisposition to develop hepatic steatosis and fibrosis, diabetes, and hypertriglyceridemia. Diagnosis of AGL is based on the observation of generalized fat loss, autoimmunity and lack of family history of lipodystrophy. The pathogenic mechanism of fat destruction remains unknown but evidences suggest an autoimmune origin. Anti-adipocyte antibodies have been previously reported in patients with AGL, although their involvement in the pathogenesis has been poorly studied and the autoantibody target/s remain/s to be identified. Using a combination of immunochemical and cellular studies, we investigated the presence of anti-adipocyte autoantibodies in patients with AGL, acquired partial lipodystrophy, localized lipoatrophy due to intradermic insulin injections or systemic lupus erythematosus. Moreover, the impact of anti-adipocyte autoantibodies from AGL patients was assessed in cultured mouse preadipocytes. Following this approach, we identified anti-perilipin 1 IgG autoantibodies in the serum of patients with autoimmune variety-AGL, but in no other lipodystrophies tested. These autoantibodies altered the ability of perilipin 1 to regulate lipolysis in cultured preadipocytes causing abnormal, significantly elevated basal lipolysis. Our data provide strong support for the conclusion that perilipin 1 autoantibodies are a cause of generalized lipodystrophy in these patients.

Originally discovered as part of C1, the initiation component of the classical complement pathway, it is now appreciated that C1q regulates a variety of cellular processes independent of complement activation. C1q is a complex glycoprotein assembled from 18 polypeptide chains, with a C-terminal globular head region that mediates recognition of diverse molecular structures, and an N-terminal collagen-like tail that mediates immune effector mechanisms. C1q mediates a variety of immunoregulatory functions considered important in the prevention of autoimmunity such as the enhancement of phagocytosis, regulation of cytokine production by antigen presenting cells, and subsequent alteration in T-lymphocyte maturation. Furthermore, recent advances indicate additional roles for C1q in diverse physiologic and pathologic processes including pregnancy, tissue repair, and cancer. Finally, C1q is emerging as a critical component of neuronal network refinement and homeostatic regulation within the central nervous system. This review summarizes the classical functions of C1q and reviews novel discoveries within the field.

The 10th C1-inhibitor deficiency workshop will be held between 18 and 21 May 2017 in Budapest (2017.haenetworkshop.hu),among the picturesque surroundings of Margaret Island.As indicated by the name of this event, most of the interest focused on angioedema due to C1-inhibitor deficiency in 1999, when it was first organized.The name is unchanged, but the range of angioedemas has expanded since to include all known varieties of hereditary and acquired angioedemas with a bradykinin-mediated pathomechanism.Looking back to the agenda of this biennial conference, many questions remained unanswered and new issues have arisen despite the enormous scientific progress made.On this occasion, 318 participants have registered from 42 countriesthis is the greatest attendance in the history of the Workshop since the start of the series.Eighty-six presentations have been submitted for this 4-day long scientific forum.The scientific program sounds interesting-it comprises novel achievements by leading scientific teams in basic research into bradykinin-mediated angioedema, the new findings of diagnostics and genetics, promising therapeutic solutions awaiting introduction, and the experience accumulated with the latest therapeutic procedures.Several presentations discuss the efforts related to improving the patients' quality of life.This time, we have invited five prominent experts-namely, Alvin Schmaier (Cleveland, OH, USA), Marco Cicardi (Milan, Italy), Avner Reshef (Tel-Hashomer, Israel), Dumitru Moldovan (Tirgu-Mures, Romania) and Attila Mócsai (Budapest, Hungary).Alvin Schmaier will show us that our knowledge about the underlying mechanisms of bradykinin-mediated angioedemas is still limited-this may be remedied by extending our interest to other forms of angioedema with different pathophysiological backgrounds.Marco Cicardi will expose the similarities and the differences between bradykinin-mediated edema formation, and the idiopathic systemic capillary leak syndrome.Avner Reshef will explore a similar issue in his presentation titled 'Angioedema-Histamine or Bradykinin?' .The lecture on neutrophil granulocytes by Attila Mócsai will take us closer to understanding the pathomechanism of angioedema.The agenda also contains the traditional roundtable session, an opportunity to develop consensus and international guidelines-this year, genetics will be in the limelight.In this session, the keynote lectures will be read by Margarita-Lopez Trascasa (Madrid, Spain) on the extended diagnostic approach integrating serological and genetic methodology; by Anastasios Germenis (Larissa, Greece) on the latest techniques for studying the SERPING1 gene; and by Nancy Brown (Nashville, TN, USA) on the pharmacogenetics of angiotensin-converting enzyme inhibitor-associated angioedema.Notwithstanding the remarkable progress made in South-America and in the former Soviet-bloc countries of Europe, state-of-the-art diagnostic and therapeutic modalities are still not available in many regions of the World.Dumitru Moldovan will review the stages along the way to making these accessible, and the experience accumulated in the effort to achieve high levels of patient care.The conference will be attended both by researchers and by clinicians-medical professionals and nurses, by the representatives of patient organizations, and by pharmaceutical industry experts involved in drug development, in order to assist the efforts of each other through joint thinking.Within the framework of this fruitful cooperation, the pharmaceutical companies also lent financial support to the conference-in addition to their scientific contribution.The travel grants, make it possible for an increasing number of professionals involved in the research or the management of patients with angioedema to attend the Workshop.The generous support by our Sponsors enabled us again to present the "For HAE Patients" award, as well as the "Grant for Young Investigators".The major donors to this event are CSL Behring and Shire.Pharming Group NV, Swedish Orphan Biovitrum, BioCryst Phamaceuticals, KininX SAS also contributed the sponsorship of the Workshop.On occasion of this tenth, jubilee event, the "For HAE Patients" award goes to Bruce Zuraw (San Diego, USA), who will present his lecture 'Let the Treatment Fit the Disease' on the festive session of the scientific section on Day 1.His achievements in bettering the management of patients will be recalled by Antony Castaldo, the chair of the International Patient Organization for C1 Inhibitor Deficiencies.The concluding event of the conference will be the awarding of the 'Grant for Young Investigators' to the top four young presenters.The support referred to above made it possible to publish the submitted abstracts of the Workshop in the journal Allergy, Asthma, and Clinical Immunology, in order to make them available to an even broader range of professionals interested in this subject.

Background: Glomerulonephritis is one of the most severe complications of lupus, a systemic disease with multi-organ involvement, with tissue damage produced mainly by complement activation. As a result of this activation, patients with active lupus present hypocomplementemia during disease flares, but C3 and C4 levels are recovered between episodes.Case presentation: We present a patient who suffered two lupus nephritis episodes in 5 years, achieving complete remission with treatment after both of them, but with C3 levels persistently below normal range. Genetic study revealed that the patient carried a mutation in heterozygosis in the C3 gene. Serial sera samples were analyzed, and autoantibodies to complement alternative pathway proteins (Factor I, Factor B, C3 and Properdin) were found. Functional assays showed that these autoantibodies cause alternative pathway activation.Conclusion: This case is the first reported of a heterozygous C3 mutation associated with lupus nephritis and autoantibodies against complement alternative pathway proteins (Factor I, Factor B, C3 and Properdin). These autoantibodies cause activation of this pathway and this fact could explain that the tissue damage is restricted to the kidney.

Atypical hemolytic uremic syndrome (aHUS) is a severe thrombotic microangiopathy affecting the renal microvasculature and is associated with complement dysregulation caused by mutations or autoanti-bodies. Disease penetrance and severity is modulated by inheritance of "risk" polymorphisms in the complement genes MCP, CFH and CFHR1. We describe the prevalence of mutations, the frequency of risk polymorphisms and the occurrence of anti-FH autoantibodies in a Spanish aHUS cohort (n=367). We also report the identification of a polymorphism in CFHR3 (c.721C>T; rs379370) that is associated with increased risk of aHUS (OR=1.78; Cl 1.22-2.59; p=0.002), and is most frequently included in an extended risk haplotype spanning the CFH-CFHR3-CFHR1 genes. This extended haplotype integrates polymorphisms in the promoter region of CFH and CFHR3, and is associated with poorer evolution of renal function and decreased FH levels. The CFH-CFHR3-CFHR1 aHUS-risk haplotype seems to be the same as was previously associated with protection against meningococcal infections, suggesting that the genetic variability in this region is limited to a few extended haplotypes, each with opposite effects in various human diseases. These results suggest that the combination of quantitative and qualitative variations in the complement proteins encoded by CFH, CFHR3 and CFHR1 genes is key for the association of these haplotypes with disease. (C) 2015 Elsevier Ltd. All rights reserved.

RecQ protein-like 4 Rothmund–Thomson syndrome Rothmund–Thomson syndrome (RTS; MIM#268400) is an autosomal recessive genodermatosis presenting in infancy with poikiloderma and heterogeneous clinical features including short stature, sparse scalp hair, sparse or absent eyelashes and/or eyebrows, juvenile cataracts, skeletal abnormalities, radial ray defects, premature aging, and a predisposition to osteosarcoma (Larizza et al., 2010Larizza L. Roversi G. Volpi L. Rothmund-Thomson syndrome.Orphanet J Rare Dis. 2010; 5: 2Crossref PubMed Scopus (189) Google Scholar). Approximately 60–65% of RTS patients carry deleterious mutations in the helicase gene RECQL4 (RTS-type II), predominantly exhibiting poikiloderma, congenital bone defects, and an increased risk of osteosarcoma. The remaining cases, characterized by poikiloderma, ectodermal dysplasia, and juvenile cataracts are negative for the RECQL4-mutation scan (RTS-type I). At the present time, the cause of RTS-type I is unknown. C1q is the central pattern-recognition molecule in the classical pathway of the complement system. Mutations causing quantitative or functional deficiency of C1q (MIM#613652) have been associated with susceptibility to severe infections, autoimmune disease frequently including systemic lupus erythematosus or overlapping clinical entities, and glomerulonephritis (Botto et al., 1998Botto M. Dell'Agnola C. Bygrave A.E. et al.Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies.Nat Genet. 1998; 19: 56-59Crossref PubMed Scopus (1243) Google Scholar; Roumenina et al., 2011Roumenina L.T. Sène D. Radanova M. et al.Functional complement C1q abnormality leads to impaired immune complexes and apoptotic cell clearance.J Immunol. 2011; 187: 4369-4373Crossref PubMed Scopus (50) Google Scholar; Schejbel et al., 2011Schejbel L. Skattum L. Hagelberg S. et al.Molecular basis of hereditary C1q deficiency—revisited: identification of several novel disease-causing mutations.Genes Immun. 2011; 12: 626-634Crossref PubMed Scopus (77) Google Scholar). Here we report novel and follow-up data on the case of a 40-year-old man belonging to an already published Spanish family (Leyva-Cobián et al., 1981Leyva-Cobián F. Moneo I. Mampaso F. et al.Familial C1q deficiency associated with renal and cutaneous disease.Clin Exp Immunol. 1981; 44: 173-180PubMed Google Scholar) in which three siblings were homozygous C1q deficient and presented with renal abnormalities and diffuse glomerular deposits of IgM and C3 compatible with mesangial proliferative glomerulonephritis. Before the age of 12 years, the two boys and a girl also developed oculocutaneus manifestations, poikiloderma, and bilateral cataracts, characteristics compatible with RTS. Both parents and three additional siblings exhibited partial C1q deficiency and remained healthy with no sign of RTS affectation. Studies have been conducted in the only survivor from the pedigree originally published by Leyva-Cobián et al., 1981Leyva-Cobián F. Moneo I. Mampaso F. et al.Familial C1q deficiency associated with renal and cutaneous disease.Clin Exp Immunol. 1981; 44: 173-180PubMed Google Scholar that provide new data on this unusual case. Clinical features in the patient at diagnosis included macular erythema, erosive skin lesions on the face and finger tips, and bilateral capsular posterior cataracts. At present, he has not developed osteosarcoma or other malignancies. To further characterize his complex phenotype, we carried out PCR amplification and direct sequencing of the C1q-A, -B, -C, and RECQL4 genes, together with a new measurement of the C1q circulating levels, which assessed total absence of the protein in plasma. Examination of the full coding and flanking intronic sequences of the C1q gene cluster on chromosome 1 revealed the homozygous, unreported c.490G>A (Gly164Ser) mutation in the third exon of the C1QC gene (Figure 1a). The glycine residue affected by this previously unreported mutation is adjacent to one of the six hydrophobic patches that define the interfaces among the A-, B-, and C-chains within the globular head (Gaboriaud et al., 2003Gaboriaud C. Juanhuix J. Gruez A. et al.The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties.J Biol Chem. 2003; 278: 46974-46982Crossref PubMed Scopus (294) Google Scholar). Therefore, the substitution of a nonpolar glycine by a bigger and more polar serine residue in such position probably has profound structural implications abolishing either multimerization or secretion of the molecule. Both parents and the remaining siblings were all heterozygous for the mutation. Western blotting of the patient’s circulating C1q with either polyclonal anti-C1q antibodies (C1q pAbs) or mAbs directed against the C1q-A (C1q-A mAbs) and -C (C1q-C mAbs) chains confirmed total absence of the full C1q molecule and also of circulating complexes between A-B and C-C chains in the plasma of the propositus (Figure 1b). No mutation was found in the 21 exons and exon–intron boundaries of the RECQL4 gene on chromosome 8, thus defining the patient as RTS-type I, which is in good agreement with his pathological condition, predominantly characterized by oculocutaneous affectation. The co-occurrence of glomerular disease and genodermatosis inherited in an apparent dominant manner is rare but not unique. The phenotypic abnormalities seen in the family presented here resemble some characteristics already reported by Sherwood et al., 1987Sherwood M.C. Pincott J.R. Goodwin F.J. et al.Dominantly inherited glomerulonephritis and an unusual skin disease.Arch Dis Child. 1987; 62: 1278-1280Crossref PubMed Scopus (16) Google Scholar in a father-and-son cohort affected by glomerulonephritis and an unusual skin disease resembling RTS. In that case, however, the activity of the classical complement pathway as measured by CH50 hemolytic assay and serum levels of C3 and C4 were all normal, discarding a homozygous defect of C1q. Studies conducted in animal models have also shown that mice lacking DNA topoisomerase III beta, an enzyme interacting with helicases of the RecQ family such as BLM (causative gene of human Bloom progeroid syndrome), develop a complex phenotype, which includes hypertrophy of the spleen and submandibular lymph nodes, glomerulonephritis, and perivascular infiltrates in various organs (Kwan and Wang, 2001Kwan K.Y. Wang J.C. Mice lacking DNA topoisomerase IIIbeta develop to maturity but show a reduced mean lifespan.Proc Natl Acad Sci USA. 2001; 98: 5717-5721Crossref PubMed Scopus (95) Google Scholar). Such a complex phenotype underscores the pleiotropic effects associated with the deregulation of DNA topological maintenance pathways and opens the possibility that genes involved in such pathways could have a role in the development of RTS-type I. With the present data, the strict co-inheritance of the C1q deficiency and RTS traits observed in this cohort suggests the presence of one or several dominant sequence variations in linkage disequilibrium with the C1q locus as a cause for the RTS-type I phenotype in the family. Alternatively, homozygous C1q deficiency could act as a disease-modifying factor for RTS in the presence of an additional, undescribed mutation in an autosomal chromosome. Written, informed consent from the patients and approval from the research ethics committee of Hospital Universitario La Paz were obtained in order to carry out these studies. We are indebted to Dr F Leyva-Cobián (Valdecilla Hospital, Santander, Spain) for his helpful discussion on the patient and his family and Drs A Torrelo (Dermatology Service) and JL Écija (Nephrology Service) from Niño Jesús Hospital (Madrid, Spain) for sharing clinical data and profitable debates on the evolution of the patient. This work was funded by the Instituto de Salud Carlos III (ISCIII) grant PS09/00122 and Ministerio de Economía y Competitividad SAF2012-38636.

Complement factor H (FH) serum levels can be affected by the presence of immune complexes of FH with autoantibodies like in autoimmune forms of atypical haemolytic uraemic syndrome (aHUS) or with C3b in homozygous factor I (FI) deficiency. These complexes reduce the amount of free functional circulating FH. In this study we aimed to determine whether FH levels measurement is disturbed in some pathological conditions and to establish a method for quantifying free and total FH in serum. For that purpose, FH levels were measured in serum samples from aHUS patients having anti-FH autoantibodies or mutations in FH gene, in patients with homozygous FI deficiency, and in healthy controls. Two anti-FH monoclonal antibodies, OX24 and A229, recognizing different functional regions in FH, were used as capture antibodies in an ELISA assay. In the control group and in the group of patients with FH mutations, the FH levels obtained with the two monoclonal antibodies were similar. In patients with anti-FH autoantibodies or with homozygous FI deficiency, however, FH levels measured with both antibodies were significantly different. As these patients had complexes of FH with autoantibodies or C3b, we interpreted that OX24 was detecting total FH and A229 was recognising free FH. Therefore, quantification of FH in plasma using these two monoclonal antibodies provides not only total FH level but also gives an estimation of how much FH circulates free and is thus available to properly control complement activation.

BACKGROUND:Hereditary Angioedema due to C1-Inhibitor deficiency (HAE types I and II) is a monogenic disease characterized by sudden, self-limited episodes of cutaneous and mucosal swelling due to local deregulation of vascular permeability. Despite its monogenic pattern of inheritance, HAE exhibits great clinical variability and low genotype/phenotype correlation among those affected, which ultimately hinders therapeutic approach and probably underlies yet unknown genetic and environmental factors.METHODS:We studied whole-genome RNA expression of PBMCs in three HAE type-I families (accounting for 40 individuals), 24 of which carry the same R472X mutation in the C1-Inhibitor gene and show large variability in terms of disease expression. Those included in this study were analyzed according to the presence of mutation and/or clinical symptoms.RESULTS:Instead of a single, common disease-associated expression pattern, we found different transcriptome signatures in two of the families studied. In one of them (referred to as DR family), symptoms correlate with the upregulation of 35 genes associated to the biological response to viral infections (including RSADs, OAS, MX and ISG pathway members) and immune response. In another pedigree (Q family), disease manifestation is linked to the upregulation of 43 genes with diverse functions, including transcription and protein folding. Moreover, symptoms-free members of the Q pedigree display relatively higher expression of 394 genes with a wide diversity of functions.CONCLUSION:We found no evidence for a common altered PBMC expression pattern linked to HAE symptoms in the three families analyzed. All the data considered, differential gene expression in PBMCs do not seem to play a significant role in the predisposition or protection against HAE in the basal -between crises- conditions analyzed. Although the RNA expression pattern associated to the response to viral infections observed in the DR family supports the idea of infectious diseases as a modifying factor for HAE severity, large-scale studies would be needed to statistically associate such expression pattern to the development of this rare disease.

Background Complement Factor I (CFI) is a serine protease with an important role in complement alternative pathway regulation. Complete factor I deficiency is strongly associated with severe infections. Approximately 30 families with this deficiency have been described worldwide. Patients and methods We have studied five new Spanish families suffering from CFI deficiency. From 19 screened people, 7 homozygous, 10 heterozygous and 2 healthy subjects were identified. Clinical, biochemical and genetic descriptions are included. Results Molecular studies demonstrated 4 novel mutations in the screened individuals; amongst them, we describe here the first great gene deletion reported in the CFI locus, which includes full exon 2 and part of the large intron 1. Conclusion CFI deficiency is possibly an underestimated defect and the eventual existence of this deficiency should be tested in those patients exhibiting low C3 and recurrent bacterial infections. We propose a simple diagnostic flowchart to help clinicians in the identification and correct diagnosis of such patients.

Hereditary angioedema due to C1 Inhibitor (C1Inh) deficiency (HAE types I and II) is a rare, life-threatening disease causing spontaneous edema of the submucosal layers. A cohort of 127 individuals with symptoms of recurrent familial angioedema from 59 non-related families was studied. All the patients included fulfilled the diagnostic and biochemical criteria of HAE, including low C1Inh function and/or concentration. Genetic studies were carried out by PCR and sequencing of the C1NH locus followed, in the negative cases, by MLPA, long-range PCR and restriction enzyme analysis of genomic DNA to detect potential large rearrangements. Mutations located in consensus splicing sequences or nearby positions were studied by RT-PCR. The study identified 52 different mutations (25 missense, 15 frameshift, 7 splicing defects and 5 large deletions) responsible for the disease in 56 HAE families. In the remaining three families no molecular alteration could be detected. Twenty-seven of the mutations in this cohort are novel and 10 are confirmed de novo cases. The pathologic effect of the 5 splicing defects first reported here was assessed at the RNA and protein levels. Large deletions affecting exons 4 and 7, ranging from approximately 1500 to 2500 bp, were partially characterized by their altered restriction patterns upon long-range amplification. These results highlight the heterogeneity of mutations in the C1NH gene causing C1Inh deficiency and HAE. An approach to the molecular effects associated to each of the mutations reported here was made when possible based on the available data of pathological variants of serpins.

To the Editor: Hereditary angioedema (HAE) caused by C1-inhibitor (C1-Inh) deficiency is a rare disease with an autosomal dominant pattern of inheritance. Clinically, HAE is characterized by episodes of localized swelling in the limbs, face, gut, or upper respiratory tract.1Grigoriadou S. Longhurst H.J. Clinical immunology review series: an approach to the patient with angio-oedema.Clin Exp Immunol. 2009; 155: 367-377Crossref PubMed Scopus (49) Google Scholar Those affected are generally heterozygous for mutations in the C1-inhibitor gene (C1NH), which result in either low levels of circulating C1-Inh (HAE type I) or normal or increased levels of a nonfunctional C1-Inh (HAE type II). Acquired forms of angioedema (AAE) caused by accelerated C1-Inh consumption associated with lymphoproliferative disorders or anti–C1-Inh autoantibodies have also been described.2Cugno M. Castelli R. Cicardi M. Angioedema due to acquired C1-Inhibitor deficiency: a bridging condition between autoimmunity and lymphoproliferation.Autoimmun Rev. 2008; 8: 156-159Crossref PubMed Scopus (88) Google Scholar HAE and AAE clinical manifestations are almost identical, but distinctive biochemical traits of patients with AAE include low amounts of C1q, C1r, and C1s proteins in serum. C1-Inh is a serum glycoprotein belonging to the serpin superfamily of protease inhibitors, the main function of which is to prevent the activation of the classical pathway of complement, as well as the activation of kinin generation, coagulation, and fibrinolytic cascades.3Cugno M. Zanichelli A. Foieni F. Caccia S. Cicardi M. C1-inhibitor deficiency and angioedema: molecular mechanisms and clinical progress.Trends Mol Med. 2009; 15: 69-78Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar Serpins are globular proteins with a conserved structure of 9 α-helices and 3 β-pleated sheets. Their reactive center is located in an exposed flexible loop referred to as the reactive center loop (RCL). They act on their target proteases by means of a suicide-substrate mechanism involving the cleavage of the RCL and its insertion into β-sheet A. As a result, conformational changes take place in the serpins that ultimately trap and inactivate the targeted protease. Nonphysiological conformations involving RCL insertion and leading to latent states, multimeric states, or both have been attributed to several naturally occurring mutations.4Eldering E. Verpy E. Roem D. Meo T. Tosi M. COOH-terminal substitutions in the serpin C1 inhibitor that cause loop overinsertion and subsequent multimerization.J Biol Chem. 1995; 270: 2579-2587Crossref PubMed Scopus (94) Google Scholar Although more than 200 different heterozygous mutations leading to HAE have been described, only 2 homozygous C1-Inh–deficient subjects, 2 siblings of consanguineous origin carrying the I440S missense mutation, have been identified. Those patients produced a nonfunctional protein and exhibited an unusual disease phenotype, with mild symptomatology and complement consumption. Moreover, they had a total absence of circulating C1q, thus resembling AAE.5Blanch A. Roche O. Urrutia I. Gamboa P. Fontán G. López-Trascasa M. First case of homozygous C1 inhibitor deficiency [published erratum in J Allergy Clin Immunol 2007;119:745].J Allergy Clin Immunol. 2006; 118: 1330-1335Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar Herein we describe a new case of homozygous C1-Inh deficiency in a patient with severe angioedema lacking circulating C1q. Genetic and functional characterization of the patient showed that he carries an R378C substitution and that this is a unique alteration resulting in partial inability of C1-Inh to control kallikrein activation. The patient is a 33-year-old man who, for the last 10 years, has experienced attacks of angioedema involving the face and extremities. He has had several episodes of laryngeal edema with ventilatory compromise. Biochemical study of the complement proteins (C1-Inh, C4, and C3 antigenic levels) was performed by means of nephelometry, C1-Inh function was determined by a specific colorimetric assay (Berichrom; Siemens, Berlin, Germany), and C1q was measured by means of radial immunodiffusion (The Binding Site, Birmingham, United Kingdom). Complement profile in the propositus revealed values concordant with a C1-Inh deficiency (C1-Inh, 4.38 mg/dL; C1-Inh function, 11%; C4, 1.44 mg/dL) and undetectable C1q, suggesting consumption of the classical pathway of complement. No lymphoproliferative disorder or anti–C1-Inh or anti-C1q autoantibodies were detected. Five additional relatives (all of them asymptomatic) were recruited for this study. Four of them had values consistent with HAE type II, and only 1 relative (GZ7; III.1), a 3-year-old girl, had C1-Inh levels and function within the normal range (see Fig E1 in this article’s Online Repository at www.jacionline.org). Once a diagnosis of HAE was established, the propositus started prophylactic treatment with the attenuated androgen stanozolol (12 mg/12 hours). Clinical examination was performed, and blood samples were obtained before treatment and at 21 and 28 months after treatment. After written informed consent was obtained, genetic studies of the C1NH locus were performed in all the family members, as previously described.5Blanch A. Roche O. Urrutia I. Gamboa P. Fontán G. López-Trascasa M. First case of homozygous C1 inhibitor deficiency [published erratum in J Allergy Clin Immunol 2007;119:745].J Allergy Clin Immunol. 2006; 118: 1330-1335Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar Sequence analysis revealed the homozygous substitution c.1198C>T (R378C) in exon 7 of the propositus. All of his relatives were heterozygous for the mutation (see Fig E1). Data from multiplex ligation-dependent probe amplification of the propositus ruled out a possible genomic deletion affecting the C1NH gene and confirmed homozygosity (data not shown). Biochemical characterization of the patient’s C1-Inh was done by means of Western blotting of fresh plasma samples. Under nonreducing conditions, native (105 kd) and inactive (cleaved or latent, 96 kd) forms of C1-Inh can be resolved. Interestingly, in the present R378C homozygote, and as reported for the other 2 previously described I440S missense homozygotes,5Blanch A. Roche O. Urrutia I. Gamboa P. Fontán G. López-Trascasa M. First case of homozygous C1 inhibitor deficiency [published erratum in J Allergy Clin Immunol 2007;119:745].J Allergy Clin Immunol. 2006; 118: 1330-1335Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar plasma C1-Inh circulates almost exclusively in its cleaved or latent state. All the heterozygous relatives studied had both forms of circulating C1-Inh (Fig 1). We studied the functionality of the R378C C1-Inh mutant by measuring complex formation with biotinylated C1s and kallikrein proteases by means of ELISA of plasma samples and supernatants of stably transfected Cos-7 cells. For the plasma assays, samples from an I440S homozygote and a patient with AAE were also analyzed. C1-Inh constructs for the Cos-7 experiments were built on the previously described C1-Inh minigene.6Vinci G. Lynch N.J. Duponchel C. Lebastard T.M. Milon G. Stover C. et al.In vivo biosynthesis of endogenous and of human C1 inhibitor in transgenic mice: tissue distribution and colocalization of their expression.J Immunol. 2002; 169: 5948-5954Crossref PubMed Scopus (12) Google Scholar A mutant clone lacking exon 8 (DelEx8) was used as a negative control for all the transfection assays. The R378C mutant protein binds C1s normally but exhibits a strongly decreased capacity to associate with kallikrein both in plasma and Cos-7 supernatants. No complexes between C1-Inh and target proteases could be detected in the I440S, AAE, and DelEx8 samples (Fig 1). This unexpected result prompted us to analyze the conformational alterations in the R378C mutant. Cleaved and latent forms of C1-Inh can be distinguished, taking advantage of the previously described, conformational-specific mAbs KII and KOK12 (kindly provided by Dr Diana Wouters, Sanquin Research, Amsterdam, The Netherlands). KII specifically recognizes cleaved inactivated C1-Inh, whereas KOK12 reacts with cleaved C1-Inh, complexed/latent C1-Inh, or both.7Nuijens J.H. Huijbregts C.C. van Mierlo G.M. Hack C.E. Inactivation of C1-inhibitor by proteases: demonstration by a monoclonal antibody of a neodeterminant on inactivated, non-complexed C1-inhibitor.Immunology. 1987; 61: 387-389PubMed Google Scholar KII signal in the homozygous R378C patient’s plasma was higher than in normal human plasma and comparable with that obtained in the I440S homozygote (an HAE type II mutation) and AAE samples. Similar detection patterns were obtained for both mutant proteins when screened with the KOK12 antibody (data not shown). The results of the I440S homozygote are consistent with complete lack of functionality because of reactive-site cleavage. In the case of the R378C homozygote and considering its normal binding to C1s, the detection of this protein by both the KII and KOK12 antibodies suggests that the R378C mutation creates a latent-like epitope recognizable by these 2 antibodies. Because only the 96-kd band was detected in this patient by means of Western blotting, we hypothesize that the C1-Inh mutant might undergo a partial insertion of the RCL into β-sheet A. One striking similarity among both the R378C and I440S families is the absence of HAE-related symptomatology in the heterozygous subjects. However, unlike the I440S homozygotes, who only reported mild clinical manifestations, the R378C homozygous patient described here did have recurrent and severe attacks of angioedema before treatment. Yet on prophylactic treatment with stanozolol, a decrease in the frequency and severity of symptoms was achieved in the R378C homozygote. This clinical amelioration coincided with a partial recovery of the patient’s complement profile. Specifically, C1-Inh, C4, and C2 concentrations were recovered to normal levels in the 2 samples analyzed after attenuated androgen therapy (posttreatment samples 1 and 2). C1q concentrations remained undetectable in posttreatment sample 1 and were only restored to normal values (10.3 mg/dL) in the second posttreatment sample (see Fig E2 in this article’s Online Repository at www.jacionline.org). A broader discussion can be found in Appendix E1 in this article’s Online Repository at www.jacionline.org. Our results further characterize the complement profile associated with homozygous mutations in the C1NH gene and suggest that suspicion of homozygous deficiency cannot rely on the presence of mild symptomatology. Additionally, we provide insights on the biochemical function of the R378C mutation, a substitution that induces a latent-like conformation of C1-Inh that specifically inhibits kallikrein binding, suggesting that the Arginine 378 position is of strategic importance for C1-Inh control of kinin formation. We thank Dr Ángeles Rico Díaz (Hospital Juan Canalejo, A Coruña, Spain) and Dr Gabriel Barderas (Centro de Salud Collado Villalba, Madrid, Spain) for referring the patients and Dr Diana Wouters (Department of Immunopathology, Sanquin Research, Amsterdam, The Netherlands) for providing the KII and KOK12 mAbs. The results in the present work are in agreement with previous reports concerning attenuated androgen treatment in patients with HAE. Androgens are known to increase C1-Inh and C4 antigenic levels in plasma of heterozygous patients with HAE.E1Gelfand J.A. Sherins R.J. Alling D.W. Frank M.M. Treatment of hereditary angioedema with danazol. Reversal of clinical and biochemical abnormalities.N Engl J Med. 1976; 295: 1444-1448Crossref PubMed Scopus (503) Google Scholar Moreover, it has been shown that androgen treatment increases C1-Inh mRNA expression in PBMCs.E2Pappalardo E. Zingale L.C. Cicardi M. Increased expression of C1-inhibitor mRNA in patients with hereditary angioedema treated with danazol.Immunol Lett. 2003; 86: 271-276Crossref PubMed Scopus (58) Google Scholar Increased levels and function of C1-Inh might restore local homeostasis between the inhibitor and its target proteases, thus controlling the activation of the classical pathway of complement in the focus of edema. Nevertheless, clinical and laboratory experience regarding androgen use in patients with HAE is restricted to heterozygous situations, in which the wild-type allele can be driven to increment C1-Inh production. To date, reports on the prophylactic intake of androgens in homozygous patients with HAE are lacking. Of note, the homozygous R378C substitution markedly reduces but does not completely suppress C1-Inh synthesis and secretion. This basal expression of the R378C allele provides a source for the low but detectable levels of C1-Inh in the patient before treatment and makes the mutant allele susceptible to androgen upregulation, explaining the patient’s good response to attenuated androgen therapy. To our knowledge, the R378C substitution described here is the first mutation outside of the RCL reported to alter C1-Inh specificity. The R378 residue is located in the gate region, in the turn between 2C helix and the S6A strand of β-sheet A (Protein Data Bank accession 2OAY). The importance of this region is highlighted by studies on the model serpin α-1-antitrypsin (A1AT), in which, on interaction with proteases, the RCL inserts into β-sheet A and is stabilized by a salt bridge formed between amino acids K290 and E342. The disruption of this salt bridge in the Z variant of A1AT (carrying the E342K mutation) reduces the stability of the molecule and induces its intracellular multimerization by means of a loop-sheet insertion mechanism.E3Janciauskiene S. Eriksson S. Callea F. Mallya M. Zhou A. Seyama K. et al.Differential detection of PAS-positive inclusions formed by the Z, Siiyama, and Mmalton variants of alpha1-antitrypsin.Hepatology. 2004; 40: 1203-1210Crossref PubMed Scopus (53) Google Scholar Based on structural models of C1-Inh, it is predictable that R378 also forms a salt bridge with E429. In the R378C mutant the positively charged arginine (homologous to K290 in A1AT) is replaced by the neutral cysteine at position 378 and probably prevents salt bridge formation with subsequent alteration of the protein conformation. The specific impairment in kallikrein activation control found in the R378C mutant sheds more light on the pathogenesis of HAE. The situation seems to be opposite to that of the family reported by Wisnieski et al,E4Wisnieski J.J. Knauss T.C. Yike I. Dearborn D.G. Narvy R.L. Naff G.B. Unique C1 inhibitor dysfunction in a kindred without angioedema, I: a mutant C1 INH that inhibits C1-s but not C1-r.J Immunol. 1994; 152: 3199-3209PubMed Google Scholar in which an Ala443Val substitution in C1-Inh prevented C1r binding but maintained full inhibitory capacity to kallikrein and coagulation factor XII.E5Zahedi R. Bissler J.J. Davis 3rd, A.E. Andreadis C. Wisnieski J.J. Unique C1 inhibitor dysfunction in a kindred without angioedema. II. Identification of an Ala443→Val substitution and functional analysis of the recombinant mutant protein.J Clin Invest. 1995; 95: 1299-1305Crossref PubMed Scopus (63) Google Scholar, E6Zahedi R. Wisnieski J.J. Davis 3rd, A.E. Role of the P2 residue of complement 1 inhibitor (Ala443) in determination of target protease specificity: inhibition of complement and contact system proteases.J Immunol. 1997; 159: 983-988PubMed Google Scholar Although the R378C homozygous patient has had recurrent episodes of edema, no family member with the Ala443Val mutation, all of whom also had complement system activation, ever had symptoms of HAE, suggesting that classical complement activation alone does not result in angioedema.

Deficiencies in complement components are rare diseases whose diagnosis is often underestimated. In addition, in only a few cases molecular studies have been carried out for the characterization of the underlying genetic defects. To date, studies involving C5-deficient patients are scarce. The aim of the present report is to characterize the biochemical and molecular complement deficiency in two non-related families with one or more members showing no detectable hemolytic complement activity (CH50<50 U/ml) and reporting a history of several episodes of meningitis. Protein deficiency was assessed by means of hemolytic assays, bi-dimensional double immunodiffusion, ELISA and Western blot of patients' sera. Molecular studies were carried out by PCR and RT-PCR of DNA and RNA, respectively, both extracted from fresh blood samples of each family member. In Family A, only the propositus had complete C5 deficiency. Molecular studies showed that he was heterozygous for two changes in the C5 gene. One of the mutations was also carried by the father (c.1883_1884AGC, Y846H) was a de novo mutation. In Family B, the two C5-deficient members share the homozygous nonsense mutation c.892C>T (Q298X) in exon 9. The characterization of these new mutations is interesting in order to elucidate structure-function relationships in the C5 gene and it also helps to understand the molecular basis of this uncommon deficiency. Moreover, this report highlights the importance of complement screening in cases of repeated meningococcal infections in order to establish its involvement and to consider adequate clinical recommendations such as prophylactic antibiotics or meningococcal vaccines.

Background To estimate the proportion of diabetic patients (DPts) with peripheral vascular disease treated at a primary health care site after an endocrinologist-based intervention, who meet ATP III and Steno targets of metabolic control, as well as to compare the outcome with the results of the patients treated by endocrinologists. Methods A controlled, prospective over 30-months period study was conducted in area 7 of Madrid. One hundred twenty six eligible diabetic patients diagnosed as having peripheral vascular disease between January 2003 and June 2004 were included in the study. After a treatment period of three months by the Diabetes team at St Carlos Hospital, 63 patients were randomly assigned to continue their follow up by diabetes team (Group A) and other 63 to be treated by the family physicians (FP) at primary care level with continuous diabetes team coordination (Group B). 57 DPts from Group A and 59 from Group B, completed the 30 months follow-up period. At baseline both groups were similar in age, weight, time from diagnosis and metabolic control. The main outcomes of this study were the proportion of patients meeting ATP III and Steno goals for HbA1c (%), Cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, blood pressure, albumine-to-creatinine excretion ratio (ACR), body mass index (BMI), waist circumference (WC), anti-aggregation treatment and smoking status. Results At the end of the follow up, no differences were found between the groups. More than 37% of diabetic patients assigned to be treated by FP achieved a HbA1c < 6.5%, more than 50% a ACR < 30 mg/g, and more than 80% reached low risk values for cholesterol, LDL cholesterol, triglycerides, diastolic blood pressure and were anti-aggregated, and 12% remained smokers. In contrast, less than 45% achieved a systolic blood pressure < 130 mm Hg, less than 12% had a BMI < 25 Kg.m-2 (versus 23% in group A; p < 0.05) and 49%/30% (men/women) had a waist circumference of low risk. Conclusion Improvements in metabolic control among diabetic patients with peripheral vascular disease treated at a primary health care setting is possible, reaching similar results to the patients treated at a specialized level. Despite such an improvement, body weight control remains more than poor in both levels, mainly at primary care level. General practitioner and endocrinologist coordination care may be important to enhance diabetes management in primary care settings. Trial registration Clinical Trial number ISRCTN75037597