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

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

Naturally occurring tumor-specific CD8(+) T-cell precursors in individuals with and without cancer

Boosting pre-existing, naturally occurring cytolytic CD8(+) T-cell (CTLs) responses directed against class-I MHC-restricted peptides of tumor antigens, represents a primary goal of cancer immunotherapy. The number of pre-existing antitumor CTLs and their impaired function has been incriminated as the most likely candidates for the reduced clinical efficacy of these trials. This study was scheduled to determine possible differences in the frequency and the function of naturally occurring CTL precursors (pCTLs) against multiple peptides derived from the cancer-testis antigens MAGE-A1 and MAGE-A3, and the overexpressed antigen hTERT, in newly diagnosed lung cancer patients as compared with aged-matched healthy individuals. The cumulative frequency of circulating peptide-specific pCTLs was found significantly higher in the cancer patients, varied widely and was not affected by radiotherapy and chemotherapy. Furthermore, this frequency was greatly different between the various tumor-antigen peptides. Under the light of recent evidence provided from animal models, these results indicate that the peptide-specific pCTL frequency might represent an important determinant for the fate of cancer immunotherapy. In addition, our results show that tumor-specific pCTLs of cancer patients can present functional differences regarding their proliferative capacity, intensity of multimer staining and lytic capacity, when compared with those of healthy individuals. Hence, our findings could have an important role for the design of improved immunotherapeutic approaches for lung cancer. Immunology and Cell Biology (2010) 88, 575-585; doi:10.1038/icb.2010.8; published online 9 February 201

Searching for genetic biomarkers for hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE)

Existing evidence indicates that modifier genes could change the phenotypic outcome of the causal SERPING1 variant and thus explain the expression variability of hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE). To further examine this hypothesis, we investigated the presence or absence of 18 functional variants of genes encoding proteins involved in the metabolism and function of bradykinin, the main mediator of C1-INH-HAE attacks, in relation to three distinct phenotypic traits of patients with C1-INH-HAE, i.e., the age at disease onset, the need for long-term prophylaxis (LTP), and the severity of the disease. Genetic analyses were performed by a validated next-generation sequencing platform. In total, 233 patients with C1-INH-HAE from 144 unrelated families from five European countries were enrolled in the study. Already described correlations between five common functional variants [F12-rs1801020, KLKB1-rs3733402, CPN1-rs61751507, and two in SERPING1 (rs4926 and rs28362944)] and C1-INH-HAE severity were confirmed. Furthermore, significant correlations were found between either the age at disease onset, the LTP, or the severity score of the disease and a series of other functional variants (F13B-rs6003, PLAU-rs2227564, SERPINA1-rs28929474, SERPINA1-rs17580, KLK1-rs5515, SERPINE1-rs6092, and F2-rs1799963). Interestingly, correlations uncovered in the entire cohort of patients were different from those discovered in the cohort of patients carrying missense causal SERPING1 variants. Our findings indicate that variants other than the SERPING1 causal variants act as independent modifiers of C1-INH-HAE severity and could be tested as possible prognostic biomarkers