A novel deep intronic "SERPING1" variant as a cause of hereditary angioedema due to C1-inhibitor deficiency

Background In about 5% of patients with hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) no mutation in the SERPING1 gene is detected. Methods C1-INH-HAE cases with no mutation in the coding region of SERPING1 after conventional genotyping were examined for defects in the intronic or untranslated regions of the gene. Using a next-generation sequencing (NGS) platform targeting the entire SERPING1, 14 unrelated C1-INH-HAE patients with no detectable mutations in the coding region of the gene were sequenced. Detected variants with a global minor allele frequency lower than the frequency of C1-INH-HAE (0.002%), were submitted to in silico analysis using ten different bioinformatics tools. Pedigree analysis and examination of their pathogenic effect on the RNA level were performed for filtered in variants. Results In two unrelated patients, the novel mutation c.-22-155G > T was detected in intron 1 of the SERPING1 gene by the use NGS and confirmed by Sanger sequencing. All bioinformatics tools predicted that the variant causes a deleterious effect on the gene and pedigree analysis showed its co-segregation with the disease. Degradation of the mutated allele was demonstrated by the loss of heterozygosity on the cDNA level. According to the American College of Medical Genetics and Genomics 2015 guidelines the c.-22-155G > T was curated as pathogenic. Conclusions For the first time, a deep intronic mutation that was detected by NGS in the SERPING1 gene, was proven pathogenic for C1-INH-HAE. Therefore, advanced DNA sequencing methods should be performed in cases of C1-INH-HAE where standard approaches fail to uncover the genetic alteration

Aspects of hereditary angioedema genotyping in the era of NGS: The case of F12 gene = Wybrane aspekty genotypowania wrodzonego obrzȩku naczynioruchowego w erze NGS: Gen F12

Objective. To screen a cohort of patients diagnosed with non-FXII angioedema for carriage of variants of F12 gene. Material and methods. DNA samples from 191 patients suffering from primary angioedema with normal C1-INH, 54 samples from non- -affected family members, and 161 samples from C1-INH-HAE (154 type I, 7 type II) patients were included in the study. The F12 gene was genotyped by targeted NGS (100% coverage of translated regions). Sanger sequencing was performed for the verification of all identified variants and family segregation studies. Results. The pathogenic F12 variant c.983C>A was detected in three patients from two unrelated families initially diagnosed as U-HAE. Six additional mutations were identified, four of which were characterized as benign (c.41T>C, c.418C>G, c.1025C>T, c.530C>T) and two of uncertain significance (c.1530G>C, c.1768T>G). Two synonymous variants (c.756C>T and c.711C>T), the common polymorphism c.619G>C, and the functional polymorphism c.-4T>C were detected in allele frequencies similar to those presented in the ExAC database for the European population. One more not yet reported synonymous variant (c. 1599A>G) was also found. Conclusion. Analyzing the entire translated region of F12 gene is important in order to identify new variants that possibly affect HAE expressivity. Interestingly, genetic analysis of F12 supports not only the diagnosis of FXII-HAE but also the correct exclusion diagnosis of U-HAE

Deciphering the genetics of primary angioedema with normal levels of C1 inhibitor

The genetic alteration underlying the great majority of primary angioedema with normal C1 inhibitor (nl-C1-INH-HAE) cases remains unknown. To search for variants associated with nl-C1-INH-HAE, we genotyped 133 unrelated nl-C1-INH-HAE patients using a custom next-generation sequencing platform targeting 55 genes possibly involved in angioedema pathogenesis. Patients already diagnosed with F12 alterations as well as those with histaminergic acquired angioedema were excluded. A variant pathogenicity curation strategy was followed, including a comparison of the results with those of genotyping 169 patients with hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE), and only filtered-in variants were studied further. Among the examined nl-C1-INH-HAE patients, carriers of neither the ANGPT1 p.Ala119Ser nor the KNG1 p.Met379Lys variant were found, whereas the PLG p.Lys330Glu was detected in four (3%) unrelated probands (one homozygote). In total, 182 different variants were curated, 21 of which represented novel mutations. Although the frequency of variants per gene was comparable between nl-C1-INH-HAE and C1-INH-HAE, variants of the KNG1 and XPNPEP1 genes were detected only in nl-C1-INH-HAE patients (six and three, respectively). Twenty-seven filtered variants in 23 different genes were detected in nl-C1-INH-HAE more than once, whereas 69/133 nl-C1-INH-HAE patients had compound heterozygotes of filtered variants located in the same or different genes. Pedigree analysis was performed where feasible. Our results indicate the role that alterations in some genes, like KNG1, may play in disease pathogenesis, the complex trait that is possibly underlying in some cases, and the existence of hitherto unrecognized disease endotypes

Cytolytic T-cell response against Epstein-Barr virus in lung cancer patients and healthy subjects

<p>Abstract</p> <p>Background</p> <p>This study aimed to examine whether EBV seropositive patients with lung cancer have an altered virus-specific CTL response, as compared to age-matched healthy controls and whether any variation in this response could be attributed to senescence.</p> <p>Methods</p> <p>Peripheral blood mononuclear cells from lung cancer patients, age-matched and younger healthy individuals were used to measure EBV-specific CTLs after in vitro amplification with the GLCTLVAML and RYSIFFDYM peptides followed by HLA-multimer staining.</p> <p>Results</p> <p>Lung cancer patients and aged-matched controls had significantly lesser EBV-specific CTL than younger healthy individuals. Multimer positive populations from either group did not differ with respect to the percentage of multimer positive CTLs and the intensity of multimer binding.</p> <p>Conclusions</p> <p>This study provides evidence that patients with lung cancer exhibit an EBV-specific CTL response equivalent to that of age-matched healthy counterparts. These data warrant the examination of whether young individuals have a more robust anti-tumor response, as is the case with the anti-EBV response.</p

Foxp3 expression in human cancer cells

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CD8 T-cell responses against MAGE antigens in lung cancer

OBJECTIVE The aim of cancer immunotherapy is to stimulate pre-existing anti-tumour specific CD8+ T cells, in order to recognise and destroy the cancer. Although such destruction has been described for a limited number of patients, the study of the interplayers of such an effect has never taken place in conjunction with normal individuals. This study was scheduled in order to determine in patients with lung cancer the magnitude and function of the pre-existing, naturally occurring cytolytic CD8+ T-cell responses against peptides of the most widely used cancer-testis antigens in cancer immunotherapy, namely MAGE-A1 and MAGE-A3 and compare these findings with those that might be unravelled in aged-matched healthy individuals.MATERIALS Peripheral blood mononuclear cells were collected from 15 patients with non-small cell lung carcinoma and 10 patients with small cell lung carcinoma upon diagnosis and 15 aged matched healthy individuals, expressing at least one of the HLA-A1, -A2, -A24 and/or HLA-B35 alleles. In 16 of the patients, cells were also collected 3-6 months post treatment with chemotherapy and/or radiotherapy. Finally, in 11 patients that underwent surgery for removal of their tumour, tumor infiltrating lymphocytes and tumor tissue were collected. METHODS The frequency of peptide-specific T cells was estimated using a sensitive method that combines HLA-multimer flow cytometric technology with a previous step of in vitro amplification under limiting dilution conditions. Amplified T cells were magnetically sorted and used to: (a) characterise their ability to lyse chromium labelled cells expressing the relevant antigens, (b) determine their TCR affinity against peptide loaded chromium labelled cells, (c) identify the expression of the differentiation molecules CD28, CD45RA, CD45RO and CCR7 by flow cytometry, (d) determine their ability to secrete IL-2, IL-4, IL-5, IL-10, IFN-γ and TNF-α after peptide specific stimulation, (e) determine the expression of Bcl-2 transcripts, and finally (f) determine the TcR Vβ sequence.RESULTS Anti-MAGE CD8 Τ cells were detected in the blood of both healthy individuals and lung cancer patients upon diagnosis. The cumulative frequency of circulating peptide-specific T cells was found to be higher in cancer patients (13±20.5 x 10-7) than normal individuals (1.8±1 x 10-7), varied widely amongst patients and was not affected by radiotherapy and/or chemotherapy. Amongst patients, the most frequent responses were those detected against MAGE-A3.Α2 and MAGE-A3.Α24 (36% και 44%, respectively), whereas 56% of normal individuals responded against the MAGE-A3.Α2 peptide. T cell responses against the other peptides were limited in both groups. Patients had similar numbers of anti-EBV T cells as did normal individuals. In none of the tumor infiltrating lymphocyte samples was an anti-MAGE T cell response detected despite the identification of anti-EBV T cells. Compared to normal individuals, CD8 T cells from patients presented with a reduced ability to lyse targets (p=0.013), proliferate to antigenic stimuli (p=0.041) and to bind the peptide-MHC complex with a high affinity (p=0.038). All anti-tumour specific T cells, irrespective of their origin, displayed a memory phenotype (CD28+/CD57-), expressed Bcl-2 and secreted ΙL-2, IL-4, IL-5, IFN-γ and TNF-α but not IL-10. CONCLUSION This study uncovered that cancer patients present with a wide variation in the frequency of peptide-specific pCTLs and this frequency differs greatly between tumor-antigen peptides. Furthermore, tumor-specific pCTLs of cancer patients present with functional differences when compared to those of healthy individuals. In the light of recent evidence, both of these findings (number and function) might have played a detrimental role in the limited success of current immunotherapy protocols and could represent an important determinant for the fate of cancer immunotherapy. Thus, improving our understanding as to what affects these parameters, promises to have a significant impact on the design of future cancer immunotherapy protocols.ΣΚΟΠΟΣ Στόχος της ανοσοθεραπείας του καρκίνου είναι η ενεργοποίηση της προϋπάρχουσας κυτταρολυτικής CD8+ Τ απάντησης έναντι αντιγόνων των όγκων με σκοπό την αναγνώριση και καταστροφή καρκινικών κυττάρων. Ενώ αυτό έχει επιτευχθεί σε ασθενείς με συγκεκριμένους τύπους καρκίνου, εντούτοις η μελέτη του φαινομένου σε πληθυσμιακό επίπεδο, καθώς και η μελέτη των χαρακτηριστικών αυτής της απάντησης με την αντίστοιχη φυσιολογικών ατόμων ιδίας ηλικίας, δεν έχει γίνει μέχρι τώρα. Ο σκοπός της παρούσας μελέτης ήταν να προσδιοριστεί σε ασθενείς με καρκίνο του πνεύμονα, κατά τη διάγνωση, το μέγεθος και τα λειτουργικά χαρακτηριστικά της προϋπάρχουσας κυτταρολυτικής CD8+ Τ απάντησης έναντι πεπτιδίων των δύο κυριοτέρων αντιγόνων των όγκων, MAGE-A1 και MAGE-A3, τα οποία χρησιμοποιούνται ευρέως στην ανοσοθεραπεία και να συγκριθούν με τα αντίστοιχα CD8+ Τ κύτταρα που ενδεχομένως ανιχνεύονται σε υγιή άτομα.ΥΛΙΚΟ Συλλέχθηκε περιφερικό αίμα από 15 ασθενείς με μη μικροκυτταρικό καρκίνο του πνεύμονα και 10 με μικροκυτταρικό καρκίνο του πνεύμονα κατά τη διάγνωση της νόσου καθώς και από 15 υγιείς μάρτυρες, οι οποίοι έφεραν τουλάχιστον ένα από τα αλλήλια HLA-A1, -A2, -A24 και/ή HLA-B35. Σε 16 από τους παραπάνω ασθενείς, περιφερικό αίμα συλλέχθηκε επίσης 3-6 μήνες μετά τη χορήγηση χημειοθεραπείας ή/και ακτινοθεραπείας. Επιπρόσθετα, από 11 από τους παραπάνω ασθενείς που υποβλήθηκαν σε χειρουργική αφαίρεση του όγκου, παρασκευάστηκε εναιώρημα κυττάρων του όγκου. ΜΕΘΟΔΟΙ Η συχνότητα των ειδικών έναντι πεπτιδίων των αντιγόνων MAGE-A1 και MAGE-A3 κυτταρολυτικών CD8+ Τ κυττάρων υπολογίστηκε χρησιμοποιώντας μία ευαίσθητη τεχνική που συνδυάζει αντιγονοειδική in vitro ενεργοποίηση υπό συνθήκες φθίνουσας συγκέντρωσης και ανίχνευση με κυτταρομετρία ροής, μέσω της τεχνολογίας των HLA-πολυμερών. Τα αντιγονοειδικά κύτταρα απομονώθηκαν με μαγνητικό διαχωρισμό και μελετήθηκε: (α) η ικανότητά τους να λύουν καρκινικά κύτταρα σεσημασμένα με 51Cr, (β) η συγγένεια του TcR τους ως προς το ειδικό πεπτίδιο με τη χρήση 51Cr σεσημασμένων κυττάρων, (γ) η έκφραση των επιφανειακών μορίων διαφοροποίησης CD28, CD45RA, CD45RO και CCR7 με κυτταρομετρία ροής, (δ) η ικανότητά τους να εκκρίνουν IL-2, IL-4, IL-5, IL-10, IFN-γ και TNF-α έπειτα από ειδική και μη ειδική πεπτιδική διέγερση, (ε) η έκφραση μεταγράφων του Bcl-2, και τέλος (ζ) η αλληλουχοποίηση της Vβ περιοχής του TCR.ΑΠΟΤΕΛΕΣΜΑΤΑ Στο περιφερικό αίμα των ασθενών, κατά τη διάγνωση, ανιχνεύθηκε αυξημένη συχνότητα αντιγονοειδικών CD8+ Τ κυττάρων που αναγνωρίζουν πεπτίδια των πρωτεϊνών MAGE-A1 και MAGE-A3 (13±20,5 x 10-7) συγκριτικά με τη συχνότητα παρόμοιων κυττάρων στο αίμα υγιών ατόμων (1,8±1 x 10-7). Η συχνότητα αυτή, παρουσίαζε μεγαλύτερη διακύμανση στους ασθενείς και δεν επηρεάστηκε από το είδος της θεραπείας. Ανάμεσα στους ασθενείς, η συχνότερη απάντηση ήταν έναντι των πεπτιδίων MAGE-A3.Α2 και MAGE-A3.Α24 (36% και 44%, αντίστοιχα), ενώ ανάμεσα στους υγιείς, το 56% εμφάνισε απάντηση έναντι του πεπτιδίου MAGE-A3.Α2. Αντίθετα, η απάντηση έναντι των MAGE-A3.A1, MAGE-A3.B35 και MAGE-A1 πεπτιδίων ήταν πρακτικά μη ανιχνεύσιμη. Οι ασθενείς με καρκίνο είχαν αντι-EBV CD8+ Τ κύτταρα στα ίδια επίπεδα με αυτά των φυσιολογικών ατόμων. Στα TILs των ασθενών, δεν ανιχνεύθηκε CD8+ Τ απάντηση έναντι πεπτιδίων των MAGE-A3 και MAGE-A1 παρά μόνο έναντι ιικών πεπτιδίων. Σε σχέση με τα φυσιολογικά άτομα, τα αντιγονοειδικά CD8+ Τ κύτταρα των ασθενών, παρουσίαζαν στατιστικά σημαντική μείωση της ικανότητάς τους να λύουν κύτταρα-στόχους (p=0,013), μειωμένη ικανότητα πολλαπλασιασμού (p=0,041) και μικρότερη ικανότητα σύνδεσης με τα HLA-πολυμερή (p=0,038). Όλα τα αντιγονοειδικά CD8+ Τ κύτταρα, ανεξαρτήτως προέλευσης, είχαν φαινότυπο μνημονικών κυττάρων (εκφράζοντας CD28, αλλά όχι CD57), εξέφραζαν Bcl-2 και εμφάνιζαν παρόμοια ικανότητα παραγωγής ΙL-2, IL-4, IL-5, IFN-γ και TNF-α, αλλά όχι IL-10. ΣΥΜΠΕΡΑΣΜΑΤΑ Παρατηρήθηκε ότι, οι ασθενείς με καρκίνο του πνεύμονα, παρουσιάζουν στο περιφερικό τους αίμα ευρύτατη διακύμανση στη συγκέντρωση αντιγονοειδικών CD8+ Τ κυττάρων με σημαντικές λειτουργικές διαφορές από τα αντίστοιχα των φυσιολογικών ατόμων. Τα χαρακτηριστικά αυτά (αριθμός και λειτουργία), θα μπορούσαν να αντιπροσωπεύουν καθοριστικούς παράγοντες για την επιτυχή έκβαση της ανοσοθεραπείας του καρκίνου και να εξηγήσουν τη μέχρι τώρα χαμηλή ανταπόκριση των ασθενών σε πρωτόκολλα εμβολιασμών

Tumor immune escape mediated by indoleamine 2,3-dioxygenase

Amongst the numerous mediators contributing towards the escape Of tumors from the host's immune response against them, the enzyme indoleamine 2,3-dioxygenase (lDO) has recently attracted special attention. By catabolizing tryptophan to N-formyl-kynurenine, IDO starves T cells from this important amino acid rendering them incapable of mounting appropriate immune responses. Originally, IDO has been associated to peripheral tolerance and maternal tolerance towards the fetus. The recent identification of IDO-expressing tumor cells has implicated this molecule as a key mediator of the tumor immune escape. Mounting evidence indicates that, within the tumor microenvironment, not only tumor cells but also other infiltrating cells such as dendritic cells, monocytes and others can be sources of IDO. IDO-induced tryptophan depletion from the tumor microenvironment could be the result of either elevated levels of the enzyme or augmented tryptophan consumption by both tumor cells and antigen presenting cells of the host. Beyond the tryptophan depletion, accumulation of its metabolites into the tumor environment seems to also propagate the suppression of anti-tumor immune responses. Finally, evidence emerges indicating that IDO possibly promotes tumor immune escape by inducing an immunoregulatory or an anergic T cell phenotype at a systemic level. In this context, anti-IDO therapeutic approaches are already under investigation, considering l-methyl-tryptophan, its analogues as well as newly identified chemicals and natural extracts. (c) 2007 Elsevier B.V. All rights reserved

Deep Intronic SERPING1 Gene Variants: Ending One Odyssey and Starting Another?

[No abstract available