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

Genetics of Hereditary Angioedema

Objective: In approximately 9.2% of patients with C1-INH-HAE, no causal variant has been identified by standard mutational screening, which is ordinarily restricted to the coding exons of the SERPING1. This suggests that, in some patients, the causative variant that modifies C1-INH expres-sion may be located in the intronic region of SERPING1. Furthermore, until now four pathogenic var-iants have been identified in exon 9 of F12 and have been associated with FXII-HAE. Of interest is the search for alterations in F12 beyond exon 9. The purpose of this study was (a) to detect pathogenic intronic variants in SERPING1 and (b) to detect new F12 variants that are potentially responsible for the FXII-HAE pathogenesis.Material and Methods: Using NGS platform targeting the entire SERPING1, 15 DNA samples be-longing to C1-INH-HAE patients with no detectable mutations in the coding region of the gene were sequenced. Of the intronic variants identified, those with frequency less than 5% were analyzed us-ing ten bioinformatics tools. Pedigree analysis and examination of their pathogenic effect on the mRNA level were performed for filtered in variants. Furthermore, DNA samples from 197 patients with nC1-INH-HAE, 161 patients with C1-INH-HAE and 46 healthy family members were genotyped by targeted NGS aiming the sequencing of the F12. Sanger sequencing was performed for the verifi-cation of all identified variants and family segregation studies.Results: Of the 37 intronic variants detected in the SERPING1, only three were further examined, c.-22-155G>T, c.890-14C>G and c.1029+384A>G. Bioinformatics tools predicted the variants as patho-genic, while pedigrees analysis showed its co-segregation with the disease. Functional study for the c.-22-155G>T variant showed degradation of the mutant allele. Moreover, six variants were identi-fied in F12, 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). Three synonymous variants (c.756C>T, c.711C>T, c.1599A>G), the common polymorphism c.619G>C and the functional polymorphism c.-4T> C were detected in allele frequencies similar to those presented in the gnomAD database for the European population. Finally, the two published pathogenic variants, c.983C> A and c.892_909dup, were detected.Conclusions: Three intronic variants were detected by NGS in the SERPING1 and were proven as pathogenic and likely 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. Furthermore, analyzing the entire translated region of F12 is important in order to identi-fy new variants that possibly affect HAE expressivity.Σκοπός: Σε περίπου 9,2% των ασθενών με κληρονομικό αγγειοοίδημα λόγω ανεπάρκειας του αναστολέα C1 εστεράσης, η παθογόνος μετάλλαξη δεν ανιχνεύεται με την αλληλούχηση των εξωνίων του SERPING1. Συμπεραίνεται ως εκ τούτου ότι η αιτιατή μετάλλαξη μπορεί να εντοπίζεται σε ιντρονική περιοχή του γονιδίου. Επιπλέον, μέχρι σήμερα έχουν ανιχνευτεί τέσσερις παθογονικές μεταλλάξεις στο εξώνιο 9 του F12 και έχουν συσχετιστεί με το FXII-HAE. Ενδιαφέρον παρουσιάζει η αναζήτηση μεταλλάξεων στο F12 πέρα από το εξώνιο 9. Κατόπιν αυτών σκοπός της μελέτης αποτέλεσε (α) η ανίχνευση παθογονικών ιντρονικών μεταλλάξεων στο SERPING1 και (β) η ανίχνευση νέων μεταλλάξεων στο γονίδιο F12 που δυνητικά ευθύνονται για την εμφάνιση του FXII-HAE.Ασθενείς και Μεθοδολογία: Χρησιμοποιώντας την τεχνολογία αλληλούχησης νέας γενεάς, η οποία στοχεύει ολόκληρο το SERPING1, προσδιορίστηκαν οι αλληλουχίες δεκαπέντε ασθενών με C1-INH-HAE χωρίς μεταλλάξεις στις εξωνικές περιοχές του γονιδίου. Από τις ιντρονικές μεταλλάξεις που εντοπίστηκαν μελετήθηκαν αυτές που είχαν συχνότητα μικρότερη από 5% και εξετάστηκαν με τη χρήση δέκα βιοπληροφορικών εργαλείων. Για τις φιλτραρισμένες μεταλλάξεις πραγματοποιήθηκαν οικογενειακές και λειτουργικές μελέτες στο επίπεδο του mRNA. Με στόχο την αλληλούχηση του γονιδίου F12, γονοτυπήθηκαν με τη πλατφόρμα NGS 197 ασθενείς με nC1-INH-HAE, 161 ασθενείς με C1-INH-HAE και 46 υγιή μέλη των οικογενειών τους. Η επαλήθευση των ανιχνευμένων μεταλλάξεων και οι οικογενειακές μελέτες πραγματοποιήθηκαν με αλληλούχηση κατά Sanger.Αποτελέσματα: Από τις 37 ιντρονικές μεταλλάξεις που ανιχνεύτηκαν στο γονίδιο SERPING1, διερευνήθηκαν περαιτέρω τρεις μεταλλάξεις, c.-22-155G>T, c.890-14C>G, c.1029+384A>G. Τα βιοπληροφορικά εργαλεία χαρακτήρισαν τις μεταλλάξεις ως παθογονικές, ενώ οι οικογενειακές μελέτες έδειξαν ότι οι μεταλλάξεις ανιχνεύονται μόνο στα ασθενή μέλη της εκάστοτε οικογένειας. Η λειτουργική μελέτη για τη μετάλλαξη c.-22-155G>T έδειξε αποικοδόμηση του μεταλλαγμένου αλληλομόρφου. Επιπλέον, στο F12 εντοπίστηκαν έξι μεταλλάξεις, τέσσερις από τις οποίες χαρακτηρίστηκαν ως καλοήθεις (c.41T>C, c.418C>G, c.1025C>T, c.530C>T) και δύο μεταλλάξεις άγνωστης παθογονικότητας (c.1530G>C, c.1768T>G). Τρεις συνώνυμες μεταλλάξεις (c.756C>T, c.711C>T, c.1599A>G), ο κοινός πολυμορφισμός c.619G>C και ο λειτουργικός πολυμορφισμός c.-4T>C ανιχνεύθηκαν σε συχνότητες αλληλομόρφων παρόμοιες με αυτές που παρουσιάζονται στη βάση gnomAD για τον ευρωπαϊκό πληθυσμό. Τέλος, ανιχνεύτηκαν οι δύο δημοσιευμένες παθογονικές μεταλλάξεις c.983C>A και c.892_909dup. Συμπεράσματα: Τρεις από τις 37 ιντρονικές μεταλλάξεις στο SERPING1 χαρακτηρίστηκαν ως παθογονικές και πιθανώς παθογονικές για το C1-INH-HAE. Επομένως, η χρήση της τεχνολογίας αλληλούχησης νέας γενεάς θα πρέπει να χρησιμοποιείται σε περιπτώσεις C1-INH-HAE, όπου οι τυπικές προσεγγίσεις αποτυγχάνουν να αποκαλύψουν τη αιτιατή μετάλλαξη. Επιπλέον, η ανάλυση ολόκληρης της μεταφραζόμενης περιοχής του F12 είναι σημαντική για την ανίχνευση νέων μεταλλάξεων που πιθανώς οδηγούν στην εμφάνιση του FXII-HAE

Wybrane aspekty genotypowania wrodzonego obrzęku 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.Cel. Przesiewowe badanie kohorty pacjentów z rozpoznanym obrzę- kiem naczynioruchowym innym niż zależny od FXII w kierunku nosicielstwa wariantów genu F12. Materiał i metody. Do badania włączono próbki DNA 191 pacjentów cierpiących na pierwotny obrzęk naczynioruchowy z prawidłowym C1-INH, 54 zdrowych członków rodzin oraz 161 pacjentów z C1-INH-HAE (154 typ I, 7 typ II). Gen F12 był genotypowany metodą NGS (obejmującą cały rejon poddany translacji). Sekwencjonowanie metodą Sangera zostało wykonane celem weryfikacji wszystkich zidentyfikowanych wariantów i badań segregacyjnych rodzin. Wyniki. U trzech pacjentów z dwóch niespokrewnionych rodzin pierwotnie zdiagnozowanych jako U-HAE wykryto patogenny wariant F12: c.983C>A. Zidentyfikowano sześć dodatkowych mutacji, z których cztery zostały określone jako łagodne (c.41T>C, c.418C>G, c.1025C>T, c.530C>T), a dwie jako mutacje o niepewnym znaczeniu (c.1530G>C, c.1768T>G). Stwierdzono dwa warianty synonimiczne (c.756C>T oraz c.711C>T), pospolity polimorfizm (c.619G>C) oraz czynnościowy polimorfizm c.-4T>C z częstością alleliczną podobną do podawanej w bazie ExAC dla populacji europejskiej. Wykryto również jeden nie raportowany dotychczas wariant synonimiczny (c. 1599A>G). Wnioski. Analiza całego rejonu genu F12 poddawanego translacji jest ważna dla identyfikowania nowych wariantów, które mogą oddziaływać na ekspresję HAE. Ponadto, analiza genetyczna F12 pozwala nie tylko na potwierdzenie rozpoznania FXII-HAE, ale również prawidłowe wykluczenie rozpoznania U-HAE