Hereditary angioedema (HAE) is a genetically determined disease characterized by recurrent attacks of edema affecting the subcutaneous and/or submucosal layers of tissue, face, lips, neck, extremities of the body,  oral cavity,  intestine and/or larynx. In the latter case, the disease becomes life-threatening.    The majority of HAE cases are associated with decreased levels of C1 (C1-esterase inhibitor), there are also descriptions of HAE with dysfunctional C1 inhibitor and HAE with normal C1 inhibitor. In the first and second variants, mutations in the C1NH gene are the cause of the disease. HAE with normal quantitative  and functional levels of C1-inhibitor has the same clinical manifestations but with mutations in other genes, including F12, PLG, ANGPT1, KNG1, MYOF, and HS3ST6. Currently, mutations in the HS3ST6 gene remain poorly understood; only one missense mutation (p.Thr144Ser, rs746467957) associated with the development of HAE has been described.The aim of our work was to study new mutations in the HS3ST6 gene and analyze in silico their prognostic nature and clinical significance for the development of hereditary angioedema.The material was whole blood samples obtained from 13 patients with symptoms of hereditary angioedema without reduced levels and function of C1-INH.Whole exome sequencing of patients, bioinformatic analysis of HS3ST6 gene mutations using a number of databases and Web resources to predict the effect of mutations on the protein and assess the conservatism of the positions of the mutations detected was involved in study methods.Mutations in the HS3ST6 gene were identified in four patients, including two cases with two mutations simultaneously. Application of bioinformatic analysis allowed us to obtain new data on four missense mutations in the studied gene. Potential pathogenetic significance was determined for three of them. The mutation NC_000016.9:g.1962132G>A (p.A163V) is most likely to be involved in pathogenesis of HAE by indirect disruption of heparan sulfate O-sulfation directly within the protein. The NC_000016.9:g.1962024G>A mutation (p.P199L) appears to lead to the development of the disease through disruption of docking with SDC2 heparan sulfate. In the NC_000016.9:g.1962046C>T (p.A192T) mutation, destabilization of the 192 amino acid position next to PAPS, may contribute to disruption of heparan sulfate O-sulfation through disruption of protein functional activity and, therefore, catalysis transfer of sulfo group to heparan sulfate syndecan-2. Thus, in all three cases, the formation of HAE appears to be possible due to disruption of the O-sulfation steps of heparan sulfate syndecan-2.Considering that in silico methods offer new opportunities to assess the pathogenetic significance of mutations, the application of bioinformatic analysis can contribute to a detailed investigation of the causes of hereditary angioedema. The present work convincingly demonstrates that rare mutations in the HS3ST6 gene may be involved in the pathogenesis of HAE and provoke edema due to increased bradykinin release.

Hereditary angioedema (HAE) is a genetically determined disorder accompanied by specific symptoms associated with sporadic subcutaneous and submucosal edema. The described cases of type III HAE not associated with mutations in the SERPING1 gene are characterized by the corresponding clinical picture with normal values and functional activity of the C1-inhibitor. Type III HAE is associated with mutations in the F12, PLG, ANGPT1, KNG1, MYOF, and HS3ST6 genes. Mutations in the MYOF and HS3ST6 genes remain the least studied as yet. And as for the MYOF gene, a single mutation, Arg217Ser, is known to be associated with the development of HAE. The purposes of this research were to study the new missense mutation NC_000010.10:g.95093020C>T in the MYOF gene and predictive assessment of its contribution to the HAE pathogenesis using the bioinformatics analysis. There was a blood sample obtained from a 14 y/o female patient with HAE clinical manifestations and without a decrease in the level and the lack in function of the C1-inhibitor. The research methods included sequencing of the patient's complete exome, bioinformatic analysis of the MYOF gene mutation using a number of databases and Internet resources with the purpose of assessing the conservation of the amino acid position of the substitution and predicting the effect of the mutation on the protein. Results: the girl had a previously undescribed missense mutation NC_000010.10:g.95093020C>T in exon 42 of the MYOF gene (isoform A) in the heterozygous state. The mutation resulted in the replacement of arginine with glutamine at position 1590 of the amino acid sequence (p.Arg1590Gln, rs201619869). The use of bioinformatics analysis allowed assuming the potential pathogenicity of the detected missense mutation, which could cause the observed edema. The possible pathways for the involvement of myoferlin with the detected mutation in the HAE pathogenesis are discussed in the Article. The use of in silico analysis allowed conducting the detailed study of the detected mutation considering its effect on the protein structure, which is the basis of its normal functioning. According to the results of the study, rare mutations in the MYOF gene can be involved in the HAE pathogenesis provoking edema through various cascades of biochemical reactions. In the course of the study, a new missense mutation in the MYOF gene, pathogenetically significant for the HAE development, was described for the first time.

The aim of this study was to characterize mutations in the hepatitis B virus (HBV) genome associated with HBsAg-negative form of the disease in patients receiving hemodialysis replacement therapy. Materials and methods. We used blood plasma samples obtained from hemodialysis centers in St. Petersburg, Russia – 173 patients and 108 patients from Belgrade, Republic of Serbia. The samples were examined for the presence of serological (HBsAg, antibodies anti-HBs IgG, anti-HBcore IgG) and molecular-genetic (HBV DNA) markers of HBV followed by whole-genome sequencing and determination of clinically significant virus mutations. Results and discussion. Antibodies to hepatitis B were detected in 7.5 % and 11.1 % of patients from St. Petersburg and Belgrade, respectively. HBsAg was identified only in 1.1 % of cases in the group from Russia and in 0.9 % of cases in the group from Serbia. HBV DNA was determined in 2.8 % of the studied samples from both, patients from Saint-Petersburg and Belgrade. Phylogenetic analysis of 9 viral isolates showed that genotype D virus (88.9 %) prevailed as compared to genotype A (11.1 %) in the examined group. Among the samples obtained from patients from St. Petersburg, four belonged to the D2 sub-genotype, one to the D3 genotype. Four samples obtained from Belgrade patients belonged to different sub-genotypes – D1, D2, D3, A2, respectively. When analyzing the nucleotide sequences of the HBV genomes, mutations in the MHR region were detected in all cases, but only in HBsAg-negative isolates, mutations were revealed in the region of 124–147 amino acids, including mutations P120T, R122K, A128V, Q129R, M133I, G145R affecting the recognition of HBsAg by anti-HBs antibodies and associated with the resistance of the virus to the vaccine. The results of this study indicate that transmission of blood-borne viral hepatitis agent in the hemodialysis departments of the Russian Federation and the Republic of Serbia still exists. The prevalence of the latent chronic hepatitis B, coupled with the presence of vaccine escape mutations in all identified cases, indicates the need to pay close attention to the occurrence of the virus mutant variants in hemodialysis centers.

Introduction. The immune status is a multifaceted parameter quantitatively and qualitatively analyzing functional activity immune system state in immune organs as well as some non-specific mechanisms of antimicrobial protection. Peripheral blood level of T-receptor excision rings (TREC) and B-cell excision rings (KREC), respectively, can serve as surrogate markers of T- and B-cell maturation. Currently, the diagnostic kits available on the market have two significant disadvantages: i) the kits are aimed at diagnosing immunodeficiency conditions only in newborns and children, while keeping adult patients uncovered; ii) essentially, use solely single reference normalization gene for data normalization resulting in increased variability and decreased sensitivity of the assay data. The aim: to develop a highly sensitive method for laboratory assessment of the state of immunity in immunodeficient patients by using real-time PCR for assessing TREC and KREC level in children and adults. Materials and methods. There were used whole blood and dry blood spot samples obtained from newborns and adults, apparently healthy individuals as well as patients with verified PID and HIV-infection. A total of 2577 samples were examined. Commercial kits were used as comparison methods. Results. Multiplex PCR was carried out, analyzing the number of target molecules TREC and KREC, as well as fragments of the HPRT and RPP30 normalization genes analyzed with the developed series of plasmid calibrators. The established analytical range of TREC/KREC DNA measurements comprised 103 to 109 copies/mL. The accuracy of measurements on a tablet-type instrument (CFX) was 95.84%, on a rotary-type instrument (Rotor-Gene 3000) 95.11%, which corresponds to the standard indicator. The equivalence between the data obtained after assessing whole blood samples and dry blood drops was shown. The data analysis allowed to find out 100%-diagnostic specificity and sensitivity of the method proposed. Conclusion. The method developed by us allows to diagnose decline in T- and/or B-cell immunity in children and adults and can be used to detect TREC and KREC molecules both in peripheral whole blood samples and dry blood spots using Guthrie cards. Moreover, the uniform values of reference norms can be used regardless of the type of analyzed clinical material. The study data evidence about potential for effective use of multiplex PCR diagnostics both for complex primary testing/screening of newborns and assessing state of immunity to identify adult patients with PID and as a part of the diagnostic monitoring of patients with secondary immunodeficiencies, e.g., HIV infection.

Increasing attention is being paid to methods for detecting primary and secondary T and/or B cell immunodeficiencies. Their implementation into laboratory diagnostics would contribute to the early diagnostics of immunodeficiencies. Currently, the number of identified adult patients with immunodeficiencies of various origins is steadily increasing. Age, gender and ethnicity of patients may be significant factors of immunity. Hence, determination of the population reference intervals for TREC and KREC DNA excision rings in peripheral blood of adult persons is an urgent laboratory task for in-depth examination of both congenital and acquired immunodeficiency conditions. Our purpose was to determine the reference intervals for the quantitative assay of TREC and KREC fragments in peripheral blood among the adult population of St. Petersburg. We studied whole blood samples obtained from 717 apparently healthy volunteers aged 18 to 108 years within the program of population immunity assessment among residents of St. Petersburg. The exclusion criterion included immunodeficiency of any origin, viral hepatitis A, B, C, HIV infection. Quantitation of the target TREC and KREC DNA fragments was carried out using a set of reagents for the quantitative determination of excisional rings TREC and KREC by Real-time PCR (TREC/KREC-AMP PS). The reference intervals were determined by the direct method according to the recommendations of the International Federation of Clinical Chemistry and the Russian State Standard (GOST) R 53022.3-2008. The volunteers were divided into six age groups: 18-29, 30-39, 40-49, 50-59, 60-69 years old, and the persons over 70. The amounts of TREC and KREC in each blood sample were determined for all age groups. Upon correlation analysis, we have revealed a negative relationship between the concentration of TREC molecules in blood samples, and the age of study participants (Spearman correlation coefficient r = -0.80 (p-value < 0.0001)). Significant differences in TREC levels between different age groups were revealed. No correlations were detected between KREC contents in blood samples and age as well as any differences between age groups. Reference intervals of the TREC level were determined for each mentioned age group. A unified reference range was established for the KREC levels. The established reference intervals for TREC and KREC molecules in adults are significantly lower than in newborns. The obtained results enable determination of reference intervals for TREC and KREC levels among adults, thus contributing to effective personalized laboratory diagnosis of immunodeficiency states of various origins.