Hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE) is a rare and life-threatening condition characterized by recurrent localized edema. We conducted a systematic screening of SERPING1 defects in a cohort of 207 Czech patients from 85 families with C1-INH-HAE. Our workflow involved a combined strategy of sequencing extended to UTR and deep intronic regions, advanced in silico prediction tools, and mRNA-based functional assays. This approach allowed us to detect a causal variant in all families except one and to identify a total of 56 different variants, including 5 novel variants that are likely to be causal. We further investigated the functional impact of two splicing variants, namely c.550 + 3A > C and c.686-7C > G using minigene assays and RT-PCR mRNA analysis. Notably, our cohort showed a considerably higher proportion of detected splicing variants compared to other central European populations and the LOVD database. Moreover, our findings revealed a significant association between HAE type 1 missense variants and a delayed HAE onset when compared to null variants. We also observed a significant correlation between the presence of the SERPING1 variant c.-21 T > C in the trans position to causal variants and the frequency of attacks per year, disease onset, as well as Clinical severity score. Overall, our study provides new insights into the genetic landscape of C1-INH-HAE in the Czech population, including the identification of novel variants and a better understanding of genotype-phenotype correlations. Our findings also highlight the importance of comprehensive screening strategies and functional analyses in improving the C1-INH-HAE diagnosis and management.

• Keywords
• C1-INH-HAE, HAE, SERPING1, genotype–phenotype relationship, hereditary angioedema, splicing, time to diagnosis,
• MeSH
• Angioedemas, Hereditary * diagnosis   epidemiology   genetics   MeSH
• Complement C1 Inhibitor Protein * genetics   MeSH
• Humans MeSH
• RNA, Messenger MeSH
• RNA Splicing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• Complement C1 Inhibitor Protein * MeSH
• RNA, Messenger MeSH
• SERPING1 protein, human MeSH Browser

Hereditary angioedema with C1 Inhibitor deficiency (C1-INH-HAE) is caused by a constellation of variants of the SERPING1 gene (n = 809; 1,494 pedigrees), accounting for 86.8% of HAE families, showing a pronounced mutagenic liability of SERPING1 and pertaining to 5.6% de novo variants. C1-INH is the major control serpin of the kallikrein-kinin system (KKS). In addition, C1-INH controls complement C1 and plasminogen activation, both systems contributing to inflammation. Recognizing the failed control of C1s protease or KKS provides the diagnosis of C1-INH-HAE. SERPING1 variants usually behave in an autosomal-dominant character with an incomplete penetrance and a low prevalence. A great majority of variants (809/893; 90.5%) that were introduced into online database have been considered as pathogenic/likely pathogenic. Haploinsufficiency is a common feature in C1-INH-HAE where a dominant-negative variant product impacts the wild-type allele and renders it inactive. Small (36.2%) and large (8.3%) deletions/duplications are common, with exon 4 as the most affected one. Point substitutions with missense variants (32.2%) are of interest for the serpin structure-function relationship. Canonical splice sites can be affected by variants within introns and exons also (14.3%). For noncanonical sequences, exon skipping has been confirmed by splicing analyses of patients' blood-derived RNAs (n = 25). Exonic variants (n = 6) can affect exon splicing. Rare deep-intron variants (n = 6), putatively acting as pseudo-exon activating mutations, have been characterized as pathogenic. Some variants have been characterized as benign/likely benign/of uncertain significance (n = 74). This category includes some homozygous (n = 10) or compound heterozygous variants (n = 11). They are presenting with minor allele frequency (MAF) below 0.00002 (i.e., lower than C1-INH-HAE frequency), and may be quantitatively unable to cause haploinsufficiency. Rare benign variants could contribute as disease modifiers. Gonadal mosaicism in C1-INH-HAE is rare and must be distinguished from a de novo variant. Situations with paternal or maternal disomy have been recorded (n = 3). Genotypes must be interpreted with biological investigation fitting with C1-INH expression and typing. Any SERPING1 variant reminiscent of the dysfunctional phenotype of serpin with multimerization or latency should be identified as serpinopathy.

• Keywords
• C1 Inhibitor, C1-INH-HAE, SERPING1 gene, angioedema, genetic variation, hereditary–diagnosis, serpin function, serpinopathy,
• Publication type
• Journal Article MeSH
• Review MeSH

Mutations can be induced by environmental factors but also arise spontaneously during DNA replication or due to deamination of methylated cytosines at CpG dinucleotides. Sites where mutations occur with higher frequency than would be expected by chance are termed hotspots while sites that contain mutations rarely are termed coldspots. Mutations are permanently scanned and repaired by repair systems. Among them, the mismatch repair targets base pair mismatches, which are discriminated from canonical base pairs by probing altered elasticity of DNA. Using biased molecular dynamics simulations, we investigated the elasticity of coldspots and hotspots motifs detected in human genes associated with inherited disorders, and also of motifs with Czech population hotspots and de novo mutations. Main attention was paid to mutations leading to G/T and A+/C pairs. We observed that hotspots without CpG/CpHpG sequences are less flexible than coldspots, which indicates that flexible sequences are more effectively repaired. In contrary, hotspots with CpG/CpHpG sequences exhibited increased flexibility as coldspots. Their mutability is more likely related to spontaneous deamination of methylated cytosines leading to C > T mutations, which are primarily targeted by base excision repair. We corroborated conclusions based on computer simulations by measuring melting curves of hotspots and coldspots containing G/T mismatch.

• Keywords
• DNA bending, Muts protein, free energy calculations, hotspots–coldspots, mutations,
• MeSH
• CpG Islands MeSH
• DNA chemistry   genetics   MeSH
• Humans MeSH
• Mutation * MeSH
• Nucleotide Motifs * MeSH
• Molecular Dynamics Simulation * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH

For more than three decades, researchers have known that consensus splice sites alone are not sufficient regulatory elements to provide complex splicing regulation. Other regulators, so-called splicing regulatory elements (SREs) are needed. Most importantly, their sequence variants often underlie the development of various human disorders. However, due to their variable location and high degeneracy, these regulatory sequences are also very difficult to recognize and predict. Many different approaches aiming to identify SREs have been tried, often leading to the development of in silico prediction tools. While these tools were initially expected to be helpful to identify splicing-affecting mutations in genetic diagnostics, we are still quite far from meeting this goal. In fact, most of these tools are not able to accurately discern the SRE-affecting pathological variants from those not affecting splicing. Nonetheless, several recent evaluations have given appealing results (namely for EX-SKIP, ESRseq and Hexplorer predictors). In this review, we aim to summarize the history of the different approaches to SRE prediction, and provide additional validation of these tools based on patients' clinical data. Finally, we evaluate their usefulness for diagnostic settings and discuss the challenges that have yet to be met.

• Keywords
• evaluation of prediction tools, in silico predictions, mutation, pre-mRNA splicing, splicing aberration, splicing regulatory elements, variants of unknown significance,
• MeSH
• Molecular Diagnostic Techniques methods   trends   MeSH
• Genetic Diseases, Inborn * MeSH
• Humans MeSH
• RNA Splice Sites * MeSH
• Mutation * MeSH
• RNA Precursors genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• RNA Splice Sites * MeSH
• RNA Precursors MeSH