Alberto López-Lera

SERPING1 mutations in 59 families with hereditary angioedema.

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.

Complement factor I deficiency: a not so rare immune defect. Characterization of new mutations and the first large gene deletion

BackgroundComplement 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 methodsWe 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.ResultsMolecular 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.ConclusionCFI 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.BACKGROUND Dystrophin is a large essential protein of skeletal and heart muscle. It is a filamentous scaffolding protein with numerous binding domains. Mutations in the DMD gene, which encodes dystrophin, mostly result in the deletion of one or several exons and cause Duchenne (DMD) and Becker (BMD) muscular dystrophies. The most common DMD mutations are frameshift mutations resulting in an absence of dystrophin from tissues. In-frame DMD mutations are less frequent and result in a protein with partial wild-type dystrophin function. The aim of this study was to highlight structural and functional modifications of dystrophin caused by in-frame mutations. METHODS AND RESULTS We developed a dedicated database for dystrophin, the eDystrophin database. It contains 209 different non frame-shifting mutations found in 945 patients from a French cohort and previous studies. Bioinformatics tools provide models of the three-dimensional structure of the protein at deletion sites, making it possible to determine whether the mutated protein retains the typical filamentous structure of dystrophin. An analysis of the structure of mutated dystrophin molecules showed that hybrid repeats were reconstituted at the deletion site in some cases. These hybrid repeats harbored the typical triple coiled-coil structure of native repeats, which may be correlated with better function in muscle cells. CONCLUSION This new database focuses on the dystrophin protein and its modification due to in-frame deletions in BMD patients. The observation of hybrid repeat reconstitution in some cases provides insight into phenotype-genotype correlations in dystrophin diseases and possible strategies for gene therapy. The eDystrophin database is freely available: http://edystrophin.genouest.org/.

C1 inhibitor function using contact-phase proteases as target: evaluation of an innovative assay

Controlling prekallikrein activation by C1 inhibitor (C1Inh) represents the most essential mechanism for angioedema patient protection. C1Inh function in the plasma is usually measured based on the residual activity of the C1s protease not involved in the pathological process. We have hereby proposed an alternative enzymatic measurement of C1Inh function based on contact‐phase activation and correlation with angioedema diagnostic requirements.

Human plasma C3 is essential for the development of memory B, but not T, lymphocytes

To the Editor: Primary C3 deficiency is an extremely rare autosomalrecessive disease, with fewer than 50 families described worldwide. Plasma and intracellular C3 are considered B-cell receptor (BCR) and T-cell receptor (TCR) costimulators, respectively, but their contribution to lymphocyte biology remains obscure, particularly in humans. Reduced plasma C3 can be caused not only by primary C3 deficiency, due to loss-of-function C3 mutations, but also by secondary C3 deficiency or C3 consumption, due to gain-of-function C3 mutations or due to mutations in C3 regulators such as complement Factor I (CFI). We reasoned that comparing Band T-cell differentiation and function in primary and secondary plasma C3 deficiency might help to understand the role of plasma and intracellular C3 in adaptive immunity. We report the immunological features of lymphocytes from 9 individuals with low plasma C3 belonging to 6 families, with mutations causing primary or secondary C3 deficiency and, in some cases, chronic kidney disease stages 1 to 3 (see Fig E1,A, and Tables E1 and E3 in this article’s Online Repository at www.jacionline.org).

Disease-modifying factors in hereditary angioedema: an RNA expression-based screening

BackgroundHereditary 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.MethodsWe 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.ResultsInstead 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.ConclusionWe 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.

Complement Study Versus CINH Gene Testing for the Diagnosis of Type I Hereditary Angioedema in Children

To the Editor, Hereditary angioedema due to C1-inhibitor deficiency (C1INH-HAE) is a rare disease inherited in an autosomal dominant manner, with reduced levels (type I) or impaired function (type II) of C1-inhibitor (C1-INH) [1]. C1-INH regulates the coagulation pathway, fibrinolytic system, complement cascade and kallikrein-kinin system. Uncontrolled activation of these pathways produces increased bradykinin levels which leads to enhanced vascular permeability and edema formation. The most common presentation involves edema of the intestinal wall causing abdominal colicky pain that may mimic an acute abdomen, and edema of the subcutaneous tissue affecting extremities, face or the genital area. Although less frequent, angioedema can also affect the larynx and cause death because of asphyxiation. An early diagnosis is therefore of vital importance. C1-INH-HAE diagnosis is usually performed by determination of antigenic concentration and functional activity of C1-INH (C1-INH, f-C1-INH) in sera/plasma. CINH gene study is also available for diagnosis, although in a few cases the mutation is not found. Most authors advise against testing complement in patients before the age of 1 year [2] because C1-INH levels had been shown to be lower than in adults [3]. However, most complement components increase during the first days, rapidly surpassing adult values [4]. To date, there are no available data comparing C1-INHHAE diagnosis carried out by means of genetic testing versus complement components determination in a pediatric population. Our aim was to assess the accuracy of complement testing in the diagnosis of type I C1-INH-HAEmade by means of genetic testing in a pediatric population. We studied twenty-nine children from twenty-one unrelated families suspected of having C1-INH-HAE due to personal and/or family history. Informed consent was obtained from parents. The Local Ethics Committee approved the study (PI-1377). Suspicion of HAE was based on personal history and/or family history of episodes of angioedema not responding to antihistamines, corticosteroids and epinephrine and/or abdominal episodes of colicky pain that did not subside with conventional therapies, as well as episodes of laryngeal oedema where other causes had been excluded. The last criterion for suspecting HAE was to have a relative who had been diagnosed with Type I C1-INH-HAE. Blood venous samples were collected. Citrated-, EDTAcoated plasma tubes and serum tubes were obtained from patients upon informed consent and approval from the local Ethics committee. C1-INH and C4 levels were quantified on serum samples by nephelometry (Siemens, Marburg, Germany) and C1-INH functionality was assayed in citrated plasma samples with the commercial kit Berichrom (Siemens) following manufacturer ’s instructions. Additionally, DNA samples were obtained from peripheral blood mononuclear cells with the Gentra Puregene BloodCore (Gentra Systems, Minneapolis, MN). Genetic analysis of suspected de novo mutations was carried out by * Maria Pedrosa m.pedrosa.d@gmail.com

Complement as a diagnostic tool in immunopathology

The complement system is a complex and autoregulated multistep cascade at the interface of innate and adaptive immunity. It is activated by immune complexes or apoptotic cells (classical pathway), pathogen-associated glycoproteins (lectin pathway) or a variety of molecular and cellular surfaces (alternative pathway). Upon activation, complement triggers the generation of proteolytic fragments that allow the elimination of the activating surface by enhancing inflammation, opsonization, phagocytosis, and cellular lysis. Moreover, complement efficiently discriminates self from non-self surfaces by means of soluble and membrane-bound complement regulators which are critical for innate self-tolerance. Complement deficiency or dysfunction disturb complement homeostasis and give rise to diseases as diverse as bacterial infections, autoimmunity, or renal and neurological disorders. Research on complement-targeted therapies is an expanding field that has already improved the prognosis of severe diseases such as atypical Haemolytic Uremic syndrome or Paroxysmal Nocturnal Haemoglobinuria. Therefore, complement analysis and monitoring provides valuable information with deep implications for diagnosis and therapy. In addition to its important role as an extracellular defense system, it has now become evident that complement is also present intracellularly, and its activation has profound implications for leukocyte survival and function. In this review, we summarize the essential, up-to-date information on the use of complement as a diagnostic and therapeutic tool in the clinics.

Expression of the SERPING1 gene is not regulated by promoter hypermethylation in peripheral blood mononuclear cells from patients with hereditary angioedema due to C1-inhibitor deficiency.

SERPING1 mutations causing Hereditary Angioedema type I (HAE-I) due to C1-Inhibitor (C1-INH) deficiency display a dominant-negative effect usually resulting in protein levels far below the expected 50%. To further investigate mechanisms for its reduced expression, we analyzed the promoter DNA methylation status of SERPING1 and its influence on C1-INH expression. Global epigenetic reactivation correlated with C1-INH mRNA synthesis and protein secretion in Huh7 hepatoma cells. However, PBMCs extracted from controls, HAE-I and HAE-II patients presented identical methylation status of the SERPING1 promoter when analyzed by bisulphite sequencing; the proximal CpG island (exon 2) is constitutively unmethylated, while the most distant one (5.7Kb upstream the transcriptional start site) is fully methylated. These results correlate with the methylation profile observed in Huh7 cells and indicate that there is not a direct epigenetic regulation of C1-INH expression in PBMCs specific for each HAE type. Other indirect modes of epigenetic regulation cannot be excluded.

Analysis of SERPING1 expression on hereditary angioedema patients: Quantitative analysis of full-length and exon 3 splicing variants

Hereditary angioedema (HAE) due to C1-inhibitor (C1-Inh) deficiency is an autosomal dominant disease caused by mutations in the SERPING1 locus. According to protein levels in plasma, two HAE phenotypes have been described: Type I, with low circulating protein levels in plasma, and Type II, where the protein is present but dysfunctional. Although more than 200 mutations have been described to date, studies on the molecular basis of this autosomic dominant trait are scarce. Previous studies demonstrated that C1-Inh mRNA expression was decreased in HAE patients. Herein, we have confirmed these findings in a large series of Spanish patients. Moreover, when our data were analyzed taking into account the type of mutation carried by the patient (i.e., missense, frameshift,…), significant differences were amongst the control, nonsense and splicing mutations groups (P<0.05). By opposite, no differences in C1-Inh mRNA expression were found between the control and HAE Type II groups, nor between treated and untreated patients groups, although a significant difference was observed between controls and untreated HAE Type I patients. An alternative splicing event has been described in the SERPING1 locus resulting in two different transcripts: the full-length and a shorter variant with skipping of exon 3. In order to investigate a possible role for this splicing in HAE, we quantified both mRNA variants in a series of 28 patients. No statistical differences were found in the expression of both variants between controls and patients when compared. However, a separate analysis considering each type of mutation evidenced a significant decrease (P: 0.0156) in the expression of the exon 3 skipping variant in those HAE Type I patients carrying nonsense mutations. Besides, median of the full variants copy number was statistically decreased on the splicing group when compared with either stop and/or missense groups. The results of these studies provide new data about C1 inhibitor expression in HAE patients and shed more light on the transcriptional regulation of the SERPING1 locus. Quantitative analysis of splicing variants could help to determine the eventual variations of these two transcripts and their possible role under inflammatory stimuli.

Targeted next-generation sequencing for the molecular diagnosis of hereditary angioedema due to C1-inhibitor deficiency

SERPING1 genotyping of subjects suspicious for hereditary angioedema due to C1-INH deficiency (C1-INH-HAE) is important for clinical practice as well as for research reasons. Conventional approaches towards the detection of C1-INH-HAE-associated SERPING1 variants are cumbersome and time-demanding with many pitfalls. To take advantage of the benefits of next-generation sequencing (NGS) technology, we developed and validated a custom NGS platform that, by targeting the entire SERPING1 gene, facilitates genetic testing of C1-INH-HAE patients in clinical practice. In total, 135 different C1-INH-HAE-associated SERPING1 variants, out of the approximately 450 reported, along with 115 negative controls and 95 randomly selected DNA samples from affected family members of C1-INH-HAE index patients, were included in the forward and reverse validation processes of this platform. Our platforms performance, i.e. analytical sensitivity of 98.96%, a false negative rate of 1.05%, analytical specificity 100%, a false positive rate equal to zero, accuracy of 99.35%, and repeatability of 100% recommends its implementation as a first line approach for the genetic testing of C1-INH-HAE patients or as a confirmatory method. A noteworthy advantage of our platform is the concomitant detection of single nucleotide variants and copy number variations throughout the whole length of the SERPING1 gene, moreover providing information about the size and the localization of the latter. During our study, 15 novel C1-INH-HAE-related SERPING1 variants were detected.