Complex analysis of the national Hereditary angioedema cohort in Slovakia - Identification of 12 novel variants in SERPING1 gene.

Background: Hereditary angioedema (HAE) is a rare autosomal dominant genetic disease characterised by acute episodes of non-pruritic skin and submucosal swelling caused by increase in vascular permeability. Objective: Here we present the first complex analysis of the National HAE Slovakian cohort with the detection of 12 previously un-published genetic variants in SERPING1 gene. Methods: In patients diagnosed with hereditary angioedema caused by deficiency or dysfunction of C1 inhibitor (C1-INH-HAE) based on clinical manifestation and complement measurements, SERPING1 gene was tested by DNA sequencing (Sanger sequencing/massive parallel sequencing) and/or multiplex ligation-dependent probe amplification for detection of large rearrangements. Results: The Slovakian national cohort consisted of 132 living patients with confirmed HAE. We identified 51 index cases (32 families, 19 sporadic patients/112 adults, 20 children). One hundred seventeen patients had HAE caused by deficiency of C1 inhibitor (C1-INH-HAE-1) and 15 patients had HAE caused by dysfunction of C1 inhibitor (C1-INH-HAE-2). The prevalence of HAE in Slovakia has recently been calculated to 1:41 280 which is higher than average calculated prevalence. The estimated incidence was 1:1360 000. Molecular-genetic testing of the SERPING1 gene found 22 unique causal variants in 26 index cases, including 12 previously undescribed and unreported. Conclusion: The first complex report about epidemiology and genetics of the Slovakian national HAE cohort expands the knowledge of the C1-INH-HAE genetics. Twelve novel causal variants were present in the half of the index cases. A higher percentage of inframe variants comparing to other studies was observed. Heterozygous deletion of exon 3 found in a large C1-INH-HAE-1 family probably causes the dysregulation of the splicing isoforms balance and leads to the decrease of full-length C1-INH level.

Systematic Approach Revealed SERPING1 Splicing-Affecting Variants to be Highly Represented in the Czech National HAE Cohort.

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.

Myeloid lineage cells evince distinct steady-state level of certain gene groups in dependence on hereditary angioedema severity.

Hereditary angioedema (HAE) is a rare genetic disorder with variable expressivity even in carriers of the same underlying genetic defect, suggesting other genetic and epigenetic factors participate in modifying HAE severity. Recent knowledge indicates the role of immune cells in several aspects of HAE pathogenesis, which makes monocytes and macrophages candidates to mediate these effects. Here we combined a search for HAE phenotype modifying gene variants with the characterization of selected genes' mRNA levels in monocyte and macrophages in a symptom-free period. While no such gene variant was found to be associated with a more severe or milder disease, patients revealed a higher number of dysregulated genes and their expression profile was significantly altered, which was typically manifested by changes in individual gene expression or by strengthened or weakened relations in mutually co-expressed gene groups, depending on HAE severity. SERPING1 showed decreased expression in HAE-C1INH patients, but this effect was significant only in patients carrying mutations supposedly activating nonsense-mediated decay. Pro-inflammatory CXC chemokine superfamily members CXCL8, 10 and 11 were downregulated, while other genes such as FCGR1A, or long non-coding RNA NEAT1 were upregulated in patients. Co-expression within some gene groups (such as an NF-kappaB function related group) was strengthened in patients with a severe and/or mild course compared to controls. All these findings show that transcript levels in myeloid cells achieve different activation or depression levels in HAE-C1INH patients than in healthy controls and/or based on disease severity and could participate in determining the HAE phenotype.

SERPING1 Variants and C1-INH Biological Function: A Close Relationship With C1-INH-HAE.

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.

A case of homozygous familial hypercholesterolemia with an atypical phenotype and delayed clinical symptoms.

We describe the casuistry of a homozygous familial hypercholesterolemia female patient with a biallelic missense variant (NM_000527.4:c.1775G>A, p.Gly592Glu) in the LDLR gene, severe hypertriglyceridemia and late manifestation of coronary heart disease not earlier than at the age of 45 years. An atypical phenotype led to a delayed diagnosis.

The Clinical and Genetic Spectrum of 82 Patients With RAG Deficiency Including a c.256_257delAA Founder Variant in Slavic Countries.

Background: Variants in recombination-activating genes (RAG) are common genetic causes of autosomal recessive forms of combined immunodeficiencies (CID) ranging from severe combined immunodeficiency (SCID), Omenn syndrome (OS), leaky SCID, and CID with granulomas and/or autoimmunity (CID-G/AI), and even milder presentation with antibody deficiency. Objective: We aim to estimate the incidence, clinical presentation, genetic variability, and treatment outcome with geographic distribution of patients with the RAG defects in populations inhabiting South, West, and East Slavic countries. Methods: Demographic, clinical, and laboratory data were collected from RAG-deficient patients of Slavic origin via chart review, retrospectively. Recombinase activity was determined in vitro by flow cytometry-based assay. Results: Based on the clinical and immunologic phenotype, our cohort of 82 patients from 68 families represented a wide spectrum of RAG deficiencies, including SCID (n = 20), OS (n = 37), and LS/CID (n = 25) phenotypes. Sixty-seven (81.7%) patients carried RAG1 and 15 patients (18.3%) carried RAG2 biallelic variants. We estimate that the minimal annual incidence of RAG deficiency in Slavic countries varies between 1 in 180,000 and 1 in 300,000 live births, and it may vary secondary to health care disparities in these regions. In our cohort, 70% (n = 47) of patients with RAG1 variants carried p.K86Vfs*33 (c.256_257delAA) allele, either in homozygous (n = 18, 27%) or in compound heterozygous (n = 29, 43%) form. The majority (77%) of patients with homozygous RAG1 p.K86Vfs*33 variant originated from Vistula watershed area in Central and Eastern Poland, and compound heterozygote cases were distributed among all Slavic countries except Bulgaria. Clinical and immunological presentation of homozygous RAG1 p.K86Vfs*33 cases was highly diverse (SCID, OS, and AS/CID) suggestive of strong influence of additional genetic and/or epigenetic factors in shaping the final phenotype. Conclusion: We propose that RAG1 p.K86Vfs*33 is a founder variant originating from the Vistula watershed region in Poland, which may explain a high proportion of homozygous cases from Central and Eastern Poland and the presence of the variant in all Slavs. Our studies in this cohort of RAG1 founder variants confirm that clinical and immunological phenotypes only partially depend on the underlying genetic defect. As access to HSCT is improving among RAG-deficient patients in Eastern Europe, we anticipate improvements in survival.

Deep Intronic Mutation in SERPING1 Caused Hereditary Angioedema Through Pseudoexon Activation.

PURPOSE: Hereditary angioedema (HAE) is a rare autosomal dominant life-threatening disease characterized by low levels of C1 inhibitor (type I HAE) or normal levels of ineffective C1 inhibitor (type II HAE), typically occurring as a consequence of a SERPING1 mutation. In some cases, a causal mutation remains undetected after using a standard molecular genetic analysis. RESULTS: Here we show a long methodological way to the final discovery of c.1029 + 384A > G, a novel deep intronic mutation in intron 6 which is responsible for HAE type I in a large family and has not been identified by a conventional diagnostic approach. This mutation results in de novo donor splice site creation and subsequent pseudoexon inclusion, the mechanism firstly described to occur in SERPING1 in this study. We additionally discovered that the proximal part of intron 6 is a region potentially prone to pseudoexon-activating mutations, since natural alternative exons and additional cryptic sites occur therein. Indeed, we confirmed the existence of at least two different alternative exons in this region not described previously. CONCLUSIONS: In conclusion, our results suggest that detecting aberrant transcripts, which are often low abundant because of nonsense-mediated decay, requires a modified methodological approach. We suggest SERPING1 intron 6 sequencing and/or tailored mRNA analysis to be routinely used in HAE patients with no mutation identified in the coding sequence.

Mendelian Susceptibility to Mycobacterial Disease: The First Case of a Diagnosed Adult Patient in the Czech Republic.

We present a case of a 42-year-old woman with Mendelian susceptibility to mycobacterial disease. The disease was diagnosed at an adult age with relatively typical clinical manifestations; the skeleton, joints, and soft tissues were affected by nontuberculous mycobacteria: Mycobacterium lentiflavum, M. kansasii, and M. avium. A previously published loss-of-function and functionally validated variant NM_000416.2:c.819_822delTAAT in IFNGR1 in a heterozygous state was detected using whole-exome sequencing. After interferon-γ therapy was started at a dose of 200 µg/m2 three times a week, there was significant clinical improvement, with the need to continue the macrolide-based combination regimen. In the last 4 months, she has been in this therapy without the need for antibiotic treatment.

Novel SAMD9 Mutation in a Patient With Immunodeficiency, Neutropenia, Impaired Anti-CMV Response, and Severe Gastrointestinal Involvement.

Mutations in the Sterile alpha motif domain containing 9 (SAMD9) gene have been described in patients with severe multisystem disorder, MIRAGE syndrome, but also in patients with bone marrow (BM) failure in the absence of other systemic symptoms. The role of hematopoietic stem cell transplantation (HSCT) in the management of the disease is still unclear. Here, we present a patient with a novel mutation in SAMD9 (c.2471 G>A, p.R824Q), manifesting with prominent gastrointestinal tract involvement and immunodeficiency, but without any sign of adrenal insufficiency typical for MIRAGE syndrome. He suffered from severe CMV (cytomegalovirus) infection at 3 months of age, with a delayed development of T lymphocyte functional response against CMV, profound T cell activation, significantly reduced B lymphocyte counts and impaired lymphocyte proliferative response. Cultured T cells displayed slightly lower calcium flux and decreased survival. At the age of 6 months, he developed severe neutropenia requiring G-CSF administration, and despite only mild morphological and immunophenotypical disturbances in the BM, 78% of the BM cells showed monosomy 7 at the age of 18 months. Surprisingly, T cell proliferation after CD3 stimulation and apoptosis of the cells normalized during the follow-up, possibly reflecting the gradual development of monosomy 7. Among other prominent symptoms, he had difficulty swallowing, requiring percutaneous endoscopic gastrostomy (PEG), frequent gastrointestinal infections, and perianal erosions. He suffered from repeated infections and periodic recurring fevers with the elevation of inflammatory markers. At 26 months of age, he underwent HSCT that significantly improved hematological and immunological laboratory parameters. Nevertheless, he continued to suffer from other conditions, and subsequently, he died at day 440 post-transplant due to sepsis. Pathogenicity of this novel SAMD9 mutation was confirmed experimentally. Expression of mutant SAMD9 caused a significant decrease in proliferation and increase in cell death of the transfected cells. Conclusion: We describe a novel SAMD9 mutation in a patient with prominent gastrointestinal and immunological symptoms but without adrenal hypoplasia. Thus, SAMD9 mutations should be considered as cause of enteropathy in pediatric patients. The insufficient therapeutic outcome of transplantation further questions the role of HSCT in the management of patients with SAMD9 mutations and multisystem involvement.

Bacterial but Not Fungal Gut Microbiota Alterations Are Associated With Common Variable Immunodeficiency (CVID) Phenotype.

Common Variable Immunodeficiency (CVID) is the most frequent symptomatic immune disorder characterized by reduced serum immunoglobulins. Patients often suffer from infectious and serious non-infectious complications which impact their life tremendously. The monogenic cause has been revealed in a minority of patients so far, indicating the role of multiple genes and environmental factors in CVID etiology. Using 16S and ITS rRNA amplicon sequencing, we analyzed the bacterial and fungal gut microbiota, respectively, in a group of 55 participants constituting of CVID patients and matched healthy controls including 16 case-control pairs living in the same household, to explore possible associations between gut microbiota composition and disease phenotype. We revealed less diverse and significantly altered bacterial but not fungal gut microbiota in CVID patients, which additionally appeared to be associated with a more severe disease phenotype. The factor of sharing the same household impacted both bacterial and fungal microbiome data significantly, although not as strongly as CVID diagnosis in bacterial assessment. Overall, our results suggest that gut bacterial microbiota is altered in CVID patients and may be one of the missing environmental drivers contributing to some of the symptoms and disease severity. Paired samples serving as controls will provide a better resolution between disease-related dysbiosis and other environmental confounders in future studies.

The Impact of DNA Extraction Methods on Stool Bacterial and Fungal Microbiota Community Recovery.

Our understanding of human gut microbiota in health and disease depends on accurate and reproducible microbial data acquisition. The critical step in this process is to apply an appropriate methodology to extract microbial DNA, since biases introduced during the DNA extraction process may result in inaccurate microbial representation. In this study, we attempted to find a DNA extraction protocol which could be effectively used to analyze both the bacterial and fungal community. We evaluated the effect of five DNA extraction methods (QIAamp DNA Stool Mini Kit, PureLinkTM Microbiome DNA Purification Kit, ZR Fecal DNA MiniPrepTM Kit, NucleoSpin® DNA Stool Kit, and IHMS protocol Q) on bacterial and fungal gut microbiome recovery using (i) a defined system of germ-free mice feces spiked with bacterial or fungal strains, and (ii) non-spiked human feces. In our experimental setup, we confirmed that the examined methods significantly differed in efficiency and quality, which affected the identified stool microbiome composition. In addition, our results indicated that fungal DNA extraction might be prone to be affected by reagent/kit contamination, and thus an appropriate blank control should be included in mycobiome research. Overall, standardized IHMS protocol Q, recommended by the International Human Microbiome Consortium, performed the best when considering all the parameters analyzed, and thus could be applied not only in bacterial, but also in fungal microbiome research.

Cost-effective genotyping of human MBL2 gene mutations using multiplex PCR.

Mannose-binding lectin (MBL) deficiency is associated with increased susceptibility to various infections and autoimmune disorders. It is caused by certain polymorphisms in the MBL2 gene promoter and mutations in the coding region of the gene. In this report, we present a novel, rapid, efficient and cost-effective method of two multiplex polymerase chain reactions (PCRs) for the assessment of three structural point mutations within exon 1 at codons 52, 54 and 57. Three additional PCR reactions for the detection of promoter polymorphisms at positions -550 and -221 were performed. MBL2 haplotypes in 359 individuals of the general Czech population were detected using this approach. The rare LYD haplotype was found in 1.1% of all alleles.