Maintenance therapy (MT), which aims to destroy the remaining tumor cells after hematopoietic stem cell transplantation (HSCT), is the final stage of treatment for acute lymphoblastic leukemia (ALL). The restoration of naive T lymphocytes is extremely important after HSCT because it provides a wide variety of T cell receptor repertoire, which is necessary to avoid recurrence of the leukemic clone. The recovery of both naive T and B lymphocytes can be demonstrated by measuring TREC/KREC-positive cells The group of patients receiving maintenance therapy after going into remission included 18 patients (male, 11; female, 7) with ALL. According to the immunophenotype ALL: common B (n = 14), cortical T cell ALL (n = 2), pre-B cell (n = 2). According to the FAB classification, 6/18 (33%) had stage L1; the remaining 12/18 (67%) had stage L2. Peripheral blood for analysis was collected from patients at the time of MT, regardless of the date of initiation of treatment; thus, the median time when the material was collected was 9 (1.2 to 15.8) months. Quantitative analysis of TREC and KREC molecules was performed by RQ-PCR. During 1.5 years of maintenance therapy in patients with ALL, the TREC count increases but does not reach normal values. B lymphocytes, in turn, recover extremely slowly and have extremely low values in all patients studied. In 5/18 patients, B cells were not detected at all. Reliable differences were found between the KREC content in patients receiving PT and healthy donors (p < 0.0001), as well as between the KREC and TREC counts in these patients. It was found that at the stage of remission induction on day 22, there is a reliable decrease and redistribution of the subpopulation composition of T and B lymphocytes, which continues until the end of consolidation. After the cancellation of high-dose chemotherapy, lymphocytes begin to gradually recover, but the rate of T lymphocytes significantly exceeds that of B lymphocytes. Quantitative analysis of TREC and KREC molecules allows us to judge the renewal of the pool of naive T and B lymphocytes without the use of additional immunological research methods. This method can be used at the stage of maintenance therapy to monitor immune reconstitution.

Janus kinase-3 (JAK3) deficiency, first described in 1995, is an autosomal recessive inborn error of immunity that mostly results in variants of severe combined immunodeficiency (SCID). The frequency is estimated to account for 7-14% of heritable SCID, with sporadic cases in the Western world. Neither preferential “hot spots” nor founder effects have yet been documented. Hereby, we aim to describe the global experience of JAK3-related diseases regarding clinical spectrum, genetic landscape, including founder variants and treatment strategies. We extracted clinical, genetic, and immunological data from published cases on patients with СID/SCID phenotype caused by defects in the JAK3 gene. The literature search included unpublished cases from collaborators, reports from meetings of the European Society for Immunodeficiencies (ESID), of the Clinical Immunology Society (CIS), and published data in the biomedical research search engine (PubMed) from 1995 to 2024. Our cohort includes 132 patients with 47 unique genetic defects, including 35 novel variants (18 homozygous, 17 heterozygous). The patients were from 5 continents with majority of Asian ancestry. Country of diagnosis included North America (USA [n = 42]); South America (Brazil [n = 3]); Europe (Turkey [n = 8], Hungary [n = 1], Poland [n = 6], UK [n = 5], Italy [n = 5], Israel [n = 4], Belarus [n = 4], Russian Federation [n = 3], Georgia [n = 3], Spain [n = 1], Germany [n = 1]); Asia (India [n = 16], Iran [n = 6],China [n = 4], Pakistan [n = 1], Japan [n = 1]); and Africa (Egypt [n = 16], Saudi Arabia [n = 1], Sudan [n = 1]). Forty-five (35%) of patients from the cohort were born to consanguineous parents in 31 families from Georgia (n = 3, 1-family), Russian Federation (n = 3/2-family), Sudan (n = 1), Turkey (n = 8, 5-family), Israel (n = 4, 2-family), India (n = 3, 2-family), Egypt(n = 13, 8-family), UK (n = 5, 4-family), China (n = 1), Brazil (n = 1), Spain (n = 1), Italy (n = 1), Pakistan (n = 1), and Saudi (n = 1). 13 novel founder variants are identified. The same founder variant was seen in several countries. The majority of genetic defects were homozygous (68%); 40/132 compound were heterozygous, and 2 patients had germline heterozygous gain-of-function. The variants occurred across the entire JAK3 gene with no hotspots. Only 6 (5%) of 132 cases developed Omenn syndrome. 61 patients were transplanted, 17 died at the age of 9 months (mean age 9-18 months), and 44 are alive. We describe for the first time a global cohort of JAK3 with 132 patients with founder effects in a subgroup. Patients were identified in four continents but are most common in countries with a high rate of consanguinity. Founder effect was identified in 14 regions.

One of the important criteria after hematopoietic stem cell transplantation (HSCT) is the reconstitution of naive lymphocytes, since they form the cellular repertoire. The number of newly formed lymphocytes after HSCT is determined by flow cytometry, but in some cases this is not always possible. As an alternative to assessing T and B cell neogenesis, determination of the number of TREC and KREC can be used. The study included 9 patients diagnosed with acute lymphoblastic leukemia aged 10.1 (4.0 to 16.1) yrs after allogeneic HSCT [HLA-matched-related (n = 4), unrelated healthy donors (n = 5)]. Monitoring points were: 30, 45, 60, 100, 180, 245, and 365 days after HSCT. Reconstitution of T and B lymphocytes was assessed based on the results of flow cytometry. Quantitative of TREC and KREC was performed using the multiplex RQ-PCR. According to the results, T-lymphocyte recovery begins 3.5 months after HSCT. TREC are detected by day 145 and by day 180 TREC is 1.8 × 103 (1.22-2.0 × 103)/106 leukocytes) without a dynamic decline up to a year after transplantation. CD3+ lymphocytes begin to appear after day 30, this picture is due to the fact that in the early post-transplant period the thymus-independent pathway of immune recovery prevails, which is mediated by donor T lymphocytes. KREC are detected from day 60 and by day 100 cross the threshold of normal values (1.8 × 103(1.7-3.1 × 103)/106 leukocytes). By day 245, KREC begins to slowly decrease, which is not a sign of transplant rejection, but indicates an increase in the total number of CD19+ lymphocytes and the effect of dilution of KREC. CD19+ lymphocytes appear by day 60 and reach the norm by day 100, this indicates that the newly formed pool of B lymphocytes mainly consists of naive cells. The recovery of naive T lymphocytes was characterized by slow dynamics up to day 100 (median 26.4%) after HSCT. Then the number of naive cells gradually increased and the recovery dynamics were similar to the recovery of TREC. By day 180 after HSCT, the median quantitative of naive T lymphocytes was 35.0%. There were no statistically significant differences in the dynamics of TREC/KREC recovery between the groups with related and unrelated transplantation. Quantitative determination of TREC/KREC allows assessment of T and B lymphocyte neogenesis after HSCT without the use of additional research methods such as flow cytometry.

Analysis of T- and B-lymphocyte recovery after hematopoietic stem cell transplantation (HSCT) is important for assessing the positive dynamics of treatment. Flow cytometry is informative enough to monitor immune recovery in the post-transplant period. However, the determination of the number of TREC and KREC can be used to assess T and B cell neogenesis. We describe 15 patients with aplastic anemia after allogeneic HSCT (HLA-matched-related [n = 8], transplantation from unrelated healthy donors [n = 7]). The age of patients was 15.0 (1.9-17.0) yrs. Monitoring points were 30, 45, 60, 100, 180, 245, 365 days after HSCT. Reconstitution of T and B lymphocytes was assessed based on the results of flow cytometry. Quantitative of TREC and KREC was performed using the multiplex RQ-PCR. KREC are detected by day 45 and reach the threshold of normal values by day 100. By day 245, the number of KREC begins to slowly decrease, which is not a sign of transplant rejection, but indicates an increase in the total number of CD19+ cells and the effect of dilution of KREC-positive lymphocytes. T-lymphocyte recovery begins later. TREC are determined by day 145 and by day 180 the median of TREC-positive lymphocytes is 800 copies per 1 × 106 lymphocytes without a dynamic decline up to a year after transplantation. In turn, according to immunophenotyping data, CD3+ begins to appear after day 30, which indicates that the number of activated T lymphocytes then decreases and the number of naive ones increases. This picture is due to the peripheral expansion of T lymphocytes, which predominate in the donor transplant, and the beginning of the production of the recipient's own cells. CD19+ lymphocytes appear by day 60 and reach the norm by day 100, which indicates that the newly formed pool of B lymphocytes mainly consists of naive cells. No statistical differences were found between the groups with related and unrelated HSCT in the dynamics of TREC reconstitution. For KREC, significant differences in the increase in quantity are observed by day 100 (p < 0.05). Quantitative determination of TREC and KREC allows assessment of T and B cell neogenesis without the use of additional research methods such as flow cytometry. This method can be used as a predictor of reconstitution of T and B cell function.

Decreased NK cell numbers or impaired NK cell function are associated with increased susceptibility to viral infections, including HSV, CMV, EBV, and VZV. In children with inborn errors of immunity (IEI), viral infections are a frequent cause of severe complications and death. This is a retrospective study of NK cell deficiency (%/absolute) in patients from Belarusian IEI registry over the past 10 years. We have found 16 patients (10 male and 6 female) with low (<5%, <100 cells/µl) NK cells. 13 are alive, one of them after HSCT; three patients died in early childhood from primary hemophagocytic lymphohistiocytosis (HLH). Most patients (58%) had mutations in genes associated with diseases of immune dysregulation PRF1, UNC13D (n = 2), SH2D1A (n = 2), XIAP, STAT3 GOF, FAS, AIRE, and NFAT5; predominantly antibody deficiency NFkB1, AICIDA, and also MYSM1, STAT3, PTPN6, and RUNX3. 6/16 had herpes viral infections, including CMV and EBV. Also, patients with low NK cell counts had clinical manifestations, such as anemia (n = 7), splenomegaly (n = 8), and lymphadenopathy (n = 3); infectious episodes in the form of bilateral pneumonia (n = 2), recurrent otitis (n = 1), and herpetic stomatitis (n = 1). Four patients had HLH. One of the patients developed Burkitt lymphoma; the other had juvenile arthritis since childhood. In our cohorts, NK cell deficiency is more common in children with diseases of immune dysregulation.

Transcobalamin II deficiency is a rare autosomal recessive disorder classified as an inborn error of immunity. Transcobalamin II is a plasma protein essential for the absorption, transport, and cellular utilization of vitamin B12. Its deficiency leads to diverse clinical manifestations, including gastrointestinal disturbances, failure to thrive, megaloblastic anemia, pancytopenia, agammaglobulinemia, neurological impairments, metabolic abnormalities, and recurrent infections. According to Orphanet and PubMed, fewer than 50 cases have been reported globally, suggesting a prevalence of less than 1 in 1,000,000. Genomic DNA was extracted from peripheral blood leukocytes using phenol-chloroform extraction. Whole-exome sequencing was performed on a DNBSEQ-G50 (MGI, China) using the Exome Capture V5 Probe Set (MGI, China). Secondary data processing of FASTQ files was performed using ZLIMS (MGI, China), and tertiary analysis involved the Annovar software, followed by variant filtering. Detected variants were evaluated using the Varsome software. Clinically significant variants were confirmed by Sanger sequencing on a 3500 Genetic Analyzer (Thermo Scientific, USA). Two novel heterozygous variants in the TCN2 gene (NM_000355.3) were identified, neither reported in the gnomAD Exomes database. These included a splice-site variant c.1223-2 A>G (VarSome classification: likely pathogenic) and a coding region variant c.154C>T, p.Pro52Ser (VarSome classification: uncertain significance). The compound heterozygous genotype was consistent with the patient’s clinical presentation. The patient was first admitted at age five with an acute anemic crisis (hemoglobin 49 g/L), required erythrocyte transfusion. Subsequent evaluations revealed persistent megaloblastic anemia, hyperbilirubinemia, and mild splenomegaly. Bone marrow analyses excluded myelodysplastic syndrome and hematologic malignancies. Iron and folate therapies were ineffective; however, cyanocobalamin normalized hemoglobin levels (130 g/L) and reduced splenomegaly. Mild intellectual impairment was noted during follow-up. Transcobalamin II deficiency typically manifests in early childhood with symptoms such as developmental delays, hypotonia, diarrhea, pallor, anemia, pancytopenia, and agammaglobulinemia. This case contributes to the understanding of the condition, expands the allelic spectrum of TCN2, and supports the optimization of treatment strategies for this rare condition.

Whole-exome sequencing (WES) has gained significant traction as a tool for both scientific research and clinical practice in diagnosing genetic disorders. Despite its high informativeness, the method is not without limitations. Moreover, target regions may exhibit incomplete coverage or lack coverage entirely. A clear understanding of the completeness of exonic region coverage in genes selected for analysis is crucial when interpreting WES data. This study included WES data from 71 patients, generated using the DNBSEQ-G50 genetic analyzer (MGI, China) and the Exome Capture V5 Probe Set (MGI, China) for DNA library preparation. Secondary data processing was performed using the ZLIMS software platform (MGI, China). The quality of sequencing results was assessed based on the exonic regions of 662 genes associated with the clinical manifestations of primary immunodeficiency. Coverage statistics were calculated using the bedtools software package (v2.27.1). Target regions for the analysis were extracted from the MGI Exome Capture V5 BED file corresponding to the Human hg19/hg37 genomic assembly. Among the genes associated with primary immunodeficiency, 339 regions within 210 genes (31% of the total gene list) exhibited coverage of fewer than 30 nucleotides. Additionally, 224 exons in 183 genes were entirely uncovered across all samples (n = 71). The absence of coverage was partially attributed to the exclusion of certain region coordinates in the BED file integrated into the ZLIMS system, which resulted in the exclusion of these regions from downstream analyses. Other contributing factors to the absence of coverage remain unresolved due to the proprietary nature of the system. Our data analysis demonstrates that the significant number of regions with low coverage necessitates careful consideration of the potential for both false-positive and false-negative results. However, even with good statistical parameters, it is important to remember the rule that the results of WES should not be used as a basis for excluding a clinical diagnosis. Our study highlights that identifying clinically significant variants may be limited not only by the inherent technical constraints of WES but also by deficiencies in the software solutions used for data processing. Even high-cost commercial data processing tools may contain critical flaws.

In children under 3 years of age, lymphoma is extremely rare and may be a manifestation of inborn errors of immunity (IEIs). The aim of this study was to determine TREC/KREC copy numbers and search for Slavic founder mutations (RAG1 p.Lys86ValfsTer33, IL7R p.Ser44Arg, NBN1 p.Lys219AsnfsTer16, ATM p.Glu1978Ter, UNC13D p.Arg782SerfsTer12) in patients with lymphoma up to 3 years of age from the Belarusian Cancer Registry (No.: 0170100025) over a 26-year period. From 1998 to 2024, 39 patients younger than 3 years (29 males and 10 females) were diagnosed with lymphoma. The median age was 2.1 years (from 50 days to 2.8 years). 4 patients had Hodgkin's lymphoma (HL), 5—diffuse large B cell lymphoma (DLBCL), 13—lymphoblastic lymphoma (LL), 10—Burkitt lymphoma (BL), 2—peripheral T cell lymphoma (PTCL), 4—anaplastic large cell lymphoma (ALCL), 1—non-Hodgkin's lymphoma (NHL) of unspecified type. DNA was isolated from archival samples from 36 patients: bone marrow smears (n = 12), frozen bone marrow cells (n = 11), and peripheral blood cells (n = 13). Lymphoma in children aged 0-3 years accounted 3% (39/1237) of all pediatric lymphoma cases up to 18 years of age, including 7% (35/481) of NHL and 0.5% (4/756) of HL patients. 15/39 (38%) patients died and 3 were lost to follow-up. TREC/KREC copy numbers was reduced in 12/36 (33%) patients, 7 of whom were dead. Low TRECs/KRECs were present in all patients with DLBCL, 2 with ALCL, 2 with LL, and single cases with HL, BL, and PTCL. Homozygosity for the underlying Slavic variant of RAG1 [p.Lys86ValfsTer33] was found in a patient with DLBCL at the age of 14 months. One 12-month-old patient with PTCL was homozygous for the founder Slavic UNC13D variant [p.Arg782SerfsTer12]. Low TREC/KREC was detected in one-third of children aged 0-3 years with lymphoma and may be used as a step 1 method to suspect the IEIs.

AIOLOS, also known as IKZF3, is a transcription factor that is highly expressed in the lymphoid lineage and is critical for lymphocyte differentiation and development. Here, we report on 9 individuals from 3 unrelated families carrying AIOLOS variants Q402* or E82K, which led to AIOLOS haploinsufficiency through different mechanisms of action. Nonsense mutant Q402* displayed abnormal DNA binding, pericentromeric targeting, posttranscriptional modification, and transcriptome regulation. Structurally, the mutant lacked the AIOLOS zinc finger (ZF) 5-6 dimerization domain, but was still able to homodimerize with WT AIOLOS and negatively regulate DNA binding through ZF1, a previously unrecognized function for this domain. Missense mutant E82K showed overall normal AIOLOS functions; however, by affecting a redefined AIOLOS protein stability domain, it also led to haploinsufficiency. Patients with AIOLOS haploinsufficiency showed hypogammaglobulinemia, recurrent infections, autoimmunity, and allergy, but with incomplete clinical penetrance. Altogether, these data redefine the AIOLOS structure-function relationship and expand the spectrum of AIOLOS-associated diseases.

Background and aimsThere is an increased risk of lymphomas in inborn errors of immunity (IEI); however, germline genetic testing is rarely used in oncological patients, even in those with early onset of cancer. Our study focuses on a child with a recombination-activating gene 1 (RAG1) deficiency who was identified through a screening program for Slavic founder genetic variants among patients who died with malignancy at an early age in Belarus.ResultsWe identified one homozygous founder RAG1 variant out of 24 available DNA samples from 71 patients who developed lymphoma aged <3 years from the Belarusian cancer registry between 1986 and 2023. Our patient had an episode of pneumonia at 3 months of age and was hospitalized for respiratory distress, candida-positive lung disease, and lymphadenopathy at 14 months of age. The diagnosis of Epstein–Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) was established. The patient had a normal lymphocyte count that decreased over time. One month after chemotherapy initiation, the patient died due to sepsis and multiple organ failure without a genetic diagnosis. In a retrospective analysis, T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) were undetectable in peripheral blood.ConclusionsA targeted screening program designed to detect a Slavic founder variant in the RAG1 gene among children revealed a 14-month-old Belarusian male infant with low TREC levels who died of EBV-driven DLBCL and complications of chemotherapy including infections. This case highlights how patients with IEI and recurrent infections may develop serious non-infectious complications, such as fatal malignancy. It also emphasizes the importance of early identification, such as newborn screening for severe combined immune deficiency. Earlier diagnosis of RAG deficiency could have prompted hematopoietic stem cell transplant well before the DLBCL occurrence. This likely would impact the onset and/or management strategies for the cancer.

Common variable immunedeficiency (CVID) is a group of diseases that are inborn errors of the immune system and are characterized by impaired production of protective antibodies. The reason is a defect in the T- and B-cells, leading to impaired ability to produce specific antibodies after vaccination and infections with a significant decrease in immunoglobulins. Laboratory methods for diagnosing CVID usually include: flow cytometry and enzyme-linked immunosorbent assay to determine the levels of immunoglobulins (IgG, IgA, IgM) and antibody titer, however, these methods do not provide sufficient information about the neogenesis of lymphocytes. Our study is aimed at obtaining information about the possibility of using a simple and effective method for determining T- and B-lymphocyte receptor recombination products (TREC/KREC) in CVID. This method allows you to evaluate the neogenesis of T and B lymphocytes. The study included 12 patients diagnosed with CVID at the age of 11,2 (7,8; 15,0) years. In 2 patients CVID was determined by defects in the NFkB1 and NFkB2 genes. In 10 patients with CVID, the genetic defect was not identified; the diagnosis was established on the basis of clinical and laboratory data. To assess the diagnostic significance of the analyzed method, ROC analysis was used, followed by calculation of diagnostic sensitivity and specificity for each indicator. Our data allows us to assert that the quantitative determination of TREC/ KREC allows using this method with high diagnostic sensitivity and specificity at the stages of diagnosing CVID.

Warts, Hypogammglobulinemia, Infections, Myelokathexis (WHIM) syndrome is a rare immunodeficiency disease that results from impaired leukocyte trafficking (myelokathexis) predominately caused by gain-of-function variants in C-X-C chemokine receptor type 4 (CXCR4). Clinical manifestations of WHIM syndrome can differ in familial forms or in people harboring identical CXCR4 variants. All known pathogenic CXCR4 variants associated with WHIM syndrome (CXCR4WHIM) to date are localized in the intracellular C-terminus of CXCR4. We identified 4 unrelated patients with variable WHIM-like clinical presentations harboring a novel heterozygous CXCR4 variant (c.250G>C; p.D84H) localized at a highly conserved position in the transmembrane domain of the receptor outside the C-terminus. Functional characterization of the CXCR4D84Hvariant (CXCR4D84H) using patient-derived peripheral blood mononuclear cells and in vitro cellular assaysshow decreased CXCR4 internalization and increased chemotaxis in response to CXCL12, similar to known CXCR4WHIM, but also revealed unique features of CXCR4D84H signaling to cAMP, Ca2+ mobilization and AKT/ERK pathways. These findings are consistent with molecular dynamics simulations that show disruption of the Na+ binding pocket by D84H, resulting in collapse of the hydrophobic gate above and destabilization of the inactive state of CXCR4. Mavorixafor, a CXCR4 antagonist being evaluated in clinical trials for chronic neutropenia and WHIM syndrome, normalized CXCL12-mediated chemotaxis of CXCR4D84H patient lymphocytes ex vivo and improved WBC and subset counts in 1 patient with CXCR4D84H enrolled in the chronic neutropenia phase 1b clinical trial (NCT04154488). The present study expands the current understanding of CXCR4 function and genotype-phenotype correlations in WHIM syndrome and in people with WHIM-like phenotypes.

Inborn immunity errors such as primary immunodeficiencies in children represent a significant problem for public health, and it is undeniably important to improve the laboratory diagnosis of this pathology by creating new, effective methods for early detection of disorders involving immune mechanisms. The ROC analysis was used to evaluate the diagnostic significance of determining the copy number of T- and B-cell receptor DNA circle fragments (TREC/KREC) by multiplex real-time PCR in patients with a genetically determined diagnosis of primary immunodeficiency. Peripheral blood DNA samples of healthy children ( n = 98) aged 0.0 (0-15.0) years, who constituted the control group, and of patients with genetically confirmed primary immunodeficiency ( n = 95) aged 7.2 (0.1-18.0) years were examined. It has been established that determining the number of T and B cell receptor rearrangement products (TREC and KREC) has a high diagnostic significance in severe combined immunodeficiency, chromosomal instability syndromes such as ataxiateleangioectasia and Niimegen syndrome, diseases associated with immune dysregulation, agammoglobulinemia. Determining TREC and KREC is not informative in immunodeficiencies with non-lymphoid cell dysfunction or disorders that do not affect T- and B-cell receptor gene rearrangement such as the Wiskott-Aldrich syndrome and the chronic granulomatous disease. Determining TREC, KREC has a high diagnostic significance and can be applied in diagnosis of congenital immunity errors associated with T- and B-cell lymphopenia.

Primary immunodeficiency states are a heterogeneous group of congenital defects of cells of the immune system, which are clinically manifested in the form of recurrent infectious and autoimmune diseases of varying severity, as well as malignant neoplasms. To determine percentile TREC/KREC indices in premature newborns with various infectious perinatal pathology. In order to determine the quantitative content of TREC/KREC copies in peripheral blood, 39 premature infants with various infectious perinatal pathology were examined, which was characterized by the presence of signs of an inflammatory process at the time of determining TREC/KREC, who were in the pediatric department for premature newborns at the RNPC "Mother and Child". The examined infants were born at the gestation period of 28–34 (IU = 32.5 (28.0–33.7)) weeks.The examined infants were born at the gestation period of 28–34 (IU = 32.5 (28.0–33.7)) weeks. Percentile values of quantitative TREC indicators (number of copies × 106 leukocytes) were: 3 percentile – 1,511.00, 10 percentile – 3,715.00, 25 percentile – 7,426.00, 50 percentile – 11,355.00, 75 percentile – 26,101.50, 90 percentile – 35,047.50, 97 the percentile is 44,582.50.Percentile values of quantitative KREC indicators (number of copies × 106 leukocytes) were: 3 percentile - 1,526.50,10 percentile - 2,620.00, 25 percentile - 3,535.00, 50 percentile - 12,251.00, 75 percentile - 24,173.00, 90 percentile - 33,130.00, 97 percentile - 73,231.00.

Currently, a high correlation of the results of flow cytometry and RQ-PCR in relation to tracking the dynamics of lymphocyte recovery after hematopoietic stem cell transplantation has been repeatedly demonstrated. At the same time, the use of molecular methods to track lymphocytic reconstitution after applying of other lymphotoxic agents has not been previously studied, in particular after applying of anti-CD20 monoclonal antibodies. The process of B-lymphocytes recovery after this therapy is assessed mainly by circulating population of B-cells. As part of standard post-therapeutic monitoring, the number of CD20+ and CD19+ cells in peripheral blood is assessed by immunophenotyping. We have studied 4 females and 2 males aged 6.5 (6–11) years. 5/6 patients were diagnosed with immune thrombocytopenic purpura (ITP) and one with autoimmune hemolytic anemia during admission. Five patients with ITP have been treated with rituximab due to failure of treatment with intravenous immunoglobulin and glucocorticosteroids. For all patients, monitoring of the B-cell immunity reconstitution after 4 injections of rituximab has revealed the same picture. First B-lymphocytes begin to be detected in the peripheral blood on the 121st day (Me). On the 293rd day (Me) after the end of therapy, the number of B cells reaches the initial values. With a significantly low level of B-lymphocytes down to total absence over a period of 136 ± 100 days, naive T-cells remain within normal values. However, when compared to the TREC level of healthy children, it was significantly lower (p = 0.01), which is associated with the use of glucocorticosteroids. When comparing the data obtained by flow cytometry (CD20+ B-lymphocytes) and RQ-PCR (number of KREC molecules), a high direct correlation of 81.8% has been traced. This demonstrates that quantitative analysis of KREC molecules can be used to monitor naive B-lymphocytes after monoclonal antibody therapy. The TREC and KREC copy number quantification method can be used when it is necessary to quantify naive T- and B-lymphocytes, and it can also be used as an additional method in parallel with flow cytometry or separately as a screening method.

With inborn errors of immunity, production of T- and B-lymphocytes is disrupted, as a result of which lymphopenia is developed, which is a life-threatening sign of a child's primary immunodeficiency. It is possible to evaluate functional activity of thymus and bone marrow for early detection of primary immunodeficiency using the TREC/KREC method. We have studied DNA from a DBS of 100 preterm infants gestating from 23.5 to 37.0 weeks by RQ-PCR. The number of TREC/KREC copies has been calculated using the formula: [1,000,000 × SQ TREC (KREC)/SQ ALB/2]. The lowest number of TREC copies has been observed in the group of preterm infants 28.0 weeks or less TRECs-mediana – 1 × 103(3 × 102–3,8 × 103copies/106leukocytes), the lowest number of KREC copies has been observed in the group of children 29–32 weeks of gestation TRECs-mediana – 4.8 x103 (3.2–8.4 x103/106leukocytes). TREC copy numbers were significantly lower for preterm infants from 23.5 to 35.0 weeks' gestation (p < 0.05) comparing to TRECs number of mature newborns. The number of KREC copies was significantly lower from 23.5 to 32.0 weeks of gestation (p < 0.05) comparing to full-term infants. Evaluation of TREC/KREC has been carried out in dynamics for very preterm infants with the lowest TREC and KREC rates upon reaching their gestational age of 37.0 weeks. With an increase in gestational age, the quantitative rate of TREC/KREC for children increased, which indicated normal neonatal lymphopoiesis and exclusion of genetically determined primary immunodeficiency. Obtaining low indicators in dynamics may indicate a violation of the bone marrow function, as well as the function of thymus, which requires an in-depth immunological study to identify genetically determined immunological disorders. TREC and KREC can be used as markers of functional activity of thymus and bone marrow for newborns.

Hereditary angioedema (HAE) is an orphan genetically determined disorder with recurrent episodes of swelling in various localization. HAE is divided into two groups: HAE with C1-inhibitor deficiency and without C1-inhibitor deficiency. The main diagnostic tests are: meas urements of levels and function of C1-inhibitor, and levels of C4. These tests are not useful in all cases, for example, in HAE without C1-inhibitor deficiency. The detection of cleaved high-molecular-weight kininogen (cHK), which, even in remission, is expected to be elevated both in patients with HAE due to C1-inhibitor deficiency and in HAE with normal C1-inhibitor is a promising biomarker. The aim of this study was to determine the clinical efficiency of the cHK detection in the diagnosis of HAE. According to the results of the measurements of cHK, the patients were divided into two groups. Group №1 (100% of cHK): 27 patients, of which 19 in the study regions had variants associated with HAE, 8 didn’t have. Group №2 (not 100% of cHK): 13 patients, of which one patient had a variant associated with HAE, 12 didn’t have. The obtained data demonstrate acceptable results of using the measurement of cleaved high-molecular-weight kininogen in laboratory practice in the diagnosis of HAE.

Hemophilia A is the most common severe bleeding disorder caused by various genetic changes in the F8 gene, leading to coagulation factor VIII deficiency. Hemophilia A is characterized by high heterogeneity of genetic defects. The severity of hemophilia A varies depending on the type of genetic defects in the F8 gene. More than 3000 unique variants of the F8 gene are associated with the hemophilia A. Approximately 30% of genetic defects occur de novo. The aim of this study is to determine the spectrum of genetic defects in the F8 gene in children with hemophilia A in Belarus. The study was approved by the Independent Ethics Committee and the Scientific Council of the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (the Republic of Belarus). The study included 98 patients with hemophilia A, who had been treated or followed up at the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (the Republic of Belarus). Patients were categorized into 3 groups based on the severity of their disease: severe (n = 82), moderate (n = 3), and mild (n = 13). Twenty (20.4%) patients had a history of inhibitors to factor VIII. For our study, we used venous blood samples. Genomic DNA was isolated from leukocyte suspension (obtained from the whole blood samples) using phenol-chloroform extraction. All severe hemophilia A patients were prescreened for intron 22 and 1 inversions in the F8 gene using inverse and multiplex polymerase chain reaction assays, respectively. Sequencing of F8 coding regions was carried out by next generation sequencing. All clinically relevant variants were confirmed by Sanger sequencing. Genetic testing revealed that 99% of the patients with hemophilia A (n = 97) had pathogenic variants in the F8 gene. Intron 22 and intron 1 inversion mutations within the F8 gene were detected in 45.1% (n = 37) and 1.2% (n = 1) patients with severe hemophilia A, respectively. Two patients had an abnormal pattern of intron 1 inversion, not previously described in the literature. A total of 48 different variants in the F8 gene were detected in 57 patients using next generation sequencing. Eleven of the 48 genetic variants identified have not been previously reported.