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Introduction. Hemophilia is an X-linked hereditary blood clotting disorder caused by insufficiency of blood clotting factor VIII or IX that affects mainly men. In extremely rare cases, the disease can be observed in women, which is most often associated with asymmetric inactivation of the X chromosome. The severity of hemophilia in women does not differ from that in men. Aim – to present a clinical observation of surgical treatment of stage 4 hemophilic arthropathy in a woman with severe hemophilia A. Main findings. Female patient T., 39 years old, was admitted to the National Medical Research Center for Hematology with a preliminary diagnosis: hereditary deficiency of factor VIII. She had an extension of the APTT to 68.8 sec, a high level of Willebrand factor activity — 222 %, and the concentration of Willebrand factor antigen of 178.1 mg/l, and a decrease in the level of factor VIII to 1.6 %. According to molecular genetic analysis, intron 22 inversion associated with severe hemophilia A was detected in the F8 gene. Throughout the patient’s life, hemarthrosis of the knee, ankle and elbow joints were observed, which led to the development of severe arthropathy of varying severity. The woman underwent total knee arthroplasty and arthrolysis of the left ankle joint. The postoperative period proceeded without complications. Hemostatic replacement therapy was performed with a recombinant factor VIII.

Introduction. Von Willebrand disease (vWD) is caused by von Willebrand factor (vWF) dysfunction resulting from pathogenic variants in the vWF gene coding the vWF protein. vWD type 2N is of particular interest, as it is characterized by almost normal vWF antigen level (Ag:vWF) and vWF loss of ability to bind FVIII and protect it from premature clearance, which leads to a low FVIII coagulation activity (FVIII:C). Therefore, the same phenotype occurs in patients with 2N type of vWD and hemophilia A.Aim — to identify patients with 2N type vWD using molecular genetic methods.Methods. Data from the medical histories of vWD patients were used. The major parameter in consideration was FVIII:C to vWF:Ag ratio, which is expected to be below 0.7 in type 2N of vWD. Pathogenic variants in exons and exon-intron junctions of the vWF gene were identified by Sanger sequencing. Due to recessive inheritance of type 2N, verification of the 2N vWD diagnosis required the identification of two pathogenic variants.Results. Three patients were considered as suffering from type 2N of vWD according to hemostasis parameters (FVIII:C/vWF:Ag < 0.7). One patient with a preliminary hemophilia A diagnosis was included after sequencing of the F8 gene, which showed no alterations, so 2N type of vWD was suspected. In all cases, sequencing of the relevant functional regions of the vWF gene led to verification of vWD type 2N. One woman (patient # 4) had a homozygous pathogenic variant p.Arg854Gln (c.2561 G>A) associated with type 2N vWD. One woman (patient # 3) was a compound heterozygote for the pathogenic variant p.Arg816Trp (c.2446 C>T) associated with type 2N and a newly described insertion c.2098_2099insG, that leads to a frameshift. The woman with FVIII:C/vWF:Ag < 0.7 (patient # 1) and the patient # 2 with preliminary hemophilia А diagnosis were both compound heterozygotes for the same combination of pathogenic variants — c.2435delC and p.Thr791Met (c.2372 C>T). Pathogenic variant p.Thr791Met is associated with type 2N, while the deletion c.2435delC should lead to allele disabling.Conclusion. Molecular methods allow more precise differentiation of type 2N from other types of vWD and hemophilia A.

Introduction. In most cases, in patients with hereditary fibrinogen deficiency, clinical manifestations are represented by bleeding of varying intensity and localization. However, the clinical picture of hereditary fibrinogen deficiency can also be represented by thrombosis.Aim — to characterize the detected mutations in fibrinogen genes and to analyze prothrombotic factors in patients with hereditary hypofibrinogenemia and thrombosis.Materials and methods. Forty-nine patients with hereditary hypofibrinogenemia were observed, of which 46 patients had no history of thrombosis and 3 patients had a confirmed history of thrombosis. These 3 patients made up the study group.Results. Heterozygous mutations were found in all 3 patients in the fibrinogen gamma chain gene (FGG), one of them had a previously undescribed deletion g.2653_2684+211del, p.(Asp167Glufs*2), which removes 32 terminal nucleotides of the fifth exon of the FGG gene and leads to the formation of a stop codon in place of amino acid 168. In two other patients, there were missense mutations c.1140T>A, p.(Cys365Ser) and c.1114A>T, p.(Asp356Val), which can determine the thrombogenic properties of the altered protein structure of fibrinogen. Other prothrombotic factors were also identified: genetic polymorphisms of low thrombotic risk, surgery, taking combined oral contraceptives.Conclusion. Hereditary fibrinogen deficiency does not play a protective role in relation to the development of thrombosis and may cause the development of thrombosis, which is associated with its multifunctional role in the hemostasis system. The pathogenesis of thrombosis in patients with hereditary hypofibrinogenemia is multifactorial and may be associated with the characteristics of the main protein defect and the coexistence of hereditary and acquired thrombotic risk factors (surgical interventions, taking combined oral contraceptives, etc.).

Introduction. Pregnancy is one of the most frequent triggers of congenital and acquired forms of thrombotic thrombocytopenic purpura (TTP).Aim — to develop tactics for the treatment of pregnant women with TTP.Results. TTP was associated with pregnancy in 55.5 % of all cases of TTP in women. In 5 pregnancies in which the diagnosis of TTP was known before pregnancy, or established in the early stages, TTP was treated throughout the pregnancy. In the congenital form, plasma transfusions were performed once every two weeks until the 20th week of pregnancy, or weekly, if thrombocytopenia < 150 × 109 /L persisted. Plasma transfusions were performed weekly after the 20th week with the goal of achieving clinical remission. With acquired TTP, glucocorticosteroids and plasma exchanges were used as treatment, in 1 case — rituximab, with the aim of achieving a clinical remission and an ADAMTS13 activity > 20 %. In this group of pregnant women, 1 caesarean section was performed and there were 4 cases of vaginal deliveries, with a total of 5 children being born with an average Apgar score of 7.5. In 7 pregnancies in which TTP fi rst manifested late, leading to a delayed diagnosis, preventive and curative measures were not carried out before delivery. In this group there were 2 abortions of pregnancy, 5 surgical deliveries, 3 cases of preeclampsia, 3 acute cerebral circulatory disorders, 1 intraabdominal bleeding, 1 case of acute renal failure, with two women undergoing mechanical ventilation. There was 1 case of antenatal fetal death, with a total of 4 children being born, who were assessed on the Apgar scale with an average of 5 points.Conclusion. Timely diagnosis as well as ongoing therapeutic and preventive measures help to avoid complications during childbirth in pregnant women with TTP.

Hemophilia A is a clotting disease caused by defects in the F8 gene. A lot of them are described and most are unique or have polyphyletic origin. We here study the origin of a pathogenic variant found in a few patients. We sequenced F8 gene for seven hemophilia A patients from the Ural region, Sverdlovskaya oblast, Russia. We constructed haplotypes for them and for 21 hemophilia A patients with other defects from the same area as a control group using four previously described X-chromosome loci associated with F8 gene. We identified a new missense variant p.(His634Arg) in seven apparently unrelated patients with mild hemophilia A from Sverdlovskaya oblast. The haplotype analysis showed that all patients share the same haplotype, absent in the other patients, suggesting a founder effect. The most recent common ancestor for the p.(His634Arg) patients is estimated to exist around the end of XVII century; however, the 95% confidence interval spans from XII to early XX century. The Ural region did not suffer from the recent bottlenecks or isolation. Therefore, the founder effect could be a natural consequence of population structuring in a relatively stable population. We identified a founder effect mutation in hemophilia A, which is a quite rare event for this disease.

The review is devoted to the description of possible mechanisms of inhibitor development in hemophilia A. Most of the focus is on the genetic factors, but attention is also paid to factors of different nature, as well as their combinations that can provoke the synthesis of inhibitory antibodies. Among the genetic determinants, special attention is paid to the type of factor VIII gene mutations, as well as functional SNPs in the protein-coding genes that regulate the immune system, which were studied by different research groups using both classical and the most modern molecular genetic methods. The type of F8 gene mutations remains the most unbiassed genetic risk factor for the inhibitor development; moreover, studies aimed at the analysis of combinations of the F8 missense mutations, causing the synthesis of antibodies, with HLA class II alleles are of particular interest. For other genetic markers, further studies are required to verify their putative role in the development of the inhibitory form of hemophilia A. Among the nongenetic factors, the inhibitor development largely depends on the type of FVIII product and the intensity of therapy. However, despite numerous studies, hemophilia inhibitors remain the most difficult treatment option of this disease. In solving this problem, the construction of algorithms for predicting the risk of inhibitor development using the integration analysis that takes into account multiple factors of both genetic and other nature and their interaction can help.

Introduction. Among the most common congenital coagulopathies are haemophilia and Von Willebrand disease. These illnesses are often mimicked by orphan hereditary coagulopathies, including combined coagulation factor V and VIII deficiency.Aim — description of a clinical presentation, hampered diagnosis and choice of haemostatic therapy in a surgical patient with combined blood coagulation factor V and VIII deficiency.Main findings. We describe a clinical case of congenital combined factor V and VIII deficiency and detail the aetiology, frequency, localisation and intensity of haemorrhages. Comorbidity and surgical indications are demonstrated to require an inter-specialty medical involvement.

Background. One of the polymorphic antigens in the ABO system is antigen A, which includes many allelic variants with different expression. Immunological methods for determining the blood group of the ABO system have limitations in their use, including due to the presence of weekly expressed antigens in humans. For the correct determination of blood group according to the ABO system, genetic typing is becoming increasingly important. 89 alleles of the ABO*A gene are known. Knowledge of ABO*A gene polymorphisms and their prevalence contributes to the prevention of errors in determining the blood group of donors and recipients.Objective: to describe variants of ABO*A gene alleles in Russians and serological characteristics of the antigens encoded by them.Materials and methods. The blood of 14,000 people was examined. The blood group was determined using anti-A, anti- Aweak, anti-B, lectin (anti-A1) and gel cards. A molecular study of ABO*A gene polymorphisms was conducted in 151 people. Polymerase chain reaction with sequence-specific primers and direct Sanger sequencing were used.Results. 7 different ABO*A alleles were detected, including the ABO*A1.01 and ABO*A1.02 alleles. In 118 individuals with a weak A antigen, the ABO*A2.01 allele was the most frequent (87.29 %). Rare alleles ABO*A2.06 (5.93 %), ABO*AW.06 (4.23 %), ABO*A2.09 (0.85 %) and ABO*Ax (1.70 %) were found. Serological characteristics of A antigens variants depending on genotypes are described, variants A1, A2, A3 and very weak A were detected. Extraagglutinins α1 were absent in all persons with weakened A antigen.Conclusion. Small or mixed agglutination with Coliclones or red blood cell stratification in the gel suggest the presence of antigen A with weakened expression. Modern molecular methods make it possible to identify rare gene alleles and genotypes. Erythrocyte genomics helps to resolve the ambiguity of the serological results allows understanding the true mechanisms of particular phenotype formation and makes a contribution to ensuring the immunological safety of blood components transfusions.

Little ground squirrel (Spermophilus pygmaeus) is a polytypic species with a wide geographic range, divided by a number of large rivers (Don, Volga, and Ural rivers). Preliminary analysis of the mtDNA control region revealed substantial genetic differentiation (7%) between populations of little ground squirrel inhabiting the right and left banks of the Volga River, suggesting that large rivers act as geographic barriers for this species. Here, we present the results of the genetic diversity analysis of little ground squirrel (52 individuals from 35 localities) across the species range. The Don and Ural rivers, unlike the Volga River, had no considerable effect on the phylogeographic structure of little ground squirrel. Geographic subdivision found in ground squirrels living to the east of the Volga River could be explained by the influence of a large paleo-river flowing along the Turgai valley during glacial periods. The Volga River divides the species into two major groups (western and eastern), both with a high level of haplotype diversity. The genetic variability indices are almost twofold higher in the western clade. The results ​​of neutrality and isolation by distance tests support a relatively recent range expansion of the eastern clade. In overall, the observed structure of genetic diversity supports the hypothesis on the European origin of S. pygmaeus with the center of species origin on the territory of Caucasian geographic region. The division of little ground squirrels into western and eastern phylogenetic lineages probably occurred under the influence of fluctuations of the Caspian Sea level, due to the formation of temporary migration corridors during the regression periods.

The article provides review of possible mechanisms of inhibitor coagulopathies, in particular of acquired hemophilia A. This pathology is an extremely rare disease occurring in 1-2 cases in 1 million per year. In the present study we provide data for two clinical cases of hemophilia A in women. These cases had different development mechanisms, although both women have a newly discovered missense mutation His2026Arg in the VIII factor gene. The matter of main interest is the description of the disease development in the patient with an acquired idiopathic hemophilia A with a possible disease occurrence due to an asymmetric X-chromosome inactivation (lyonization). In this particular case lyonization led to the late manifestation of the hemophilia A carrier’s state and development of severe form of the inhibitor-associated acquired hemophilia A. We also discuss therapeutic approaches to these forms of the disease, considering there are no concise protocols for case management due to an extreme rarity of the pathology. Acquainting the clinical personnel working it the different areas of medicine with suchlike inhibitor coagulopathies has a major practical importance.

Hemophilia A is a recessive X-linked hereditary disease, so its manifestation in women is extremely rare and can be a result of an asymmetric X-chromosome inactivation or, even more rarely, of a presence of mutations in both FVIII gene alleles. We conducted a mutation screening of the FVIII gene in two female patients with clinical hemophilia A manifestation in this study. One patient had a hereditary disease; the second one was diagnosed with acquired hemophilia A as an adult. Both patients carried the same missense mutation His2026Arg. The patient with the hereditary form of the disease also had previously known microinsertion c.4379_4380 insA (p.Asn1460Lys-fs*1). We found no additional aberrations by sequencing of all functionally significant parts of the factor VIII gene of the patient with acquired hemophilia but showed clear asymmetric inactivation of X-chromosomes. Therefore, one of the possible explanations for the emergence of the hemophilic syndrome in this case can be the delayed manifestation of the FVIII gene germline mutation owing to the enhancement of hematopoiesis clonality with age.

Introduction. The group of genes, encoding cancer-testis antigens is actively studied as a new markers of refractory in some diseases, including multiple myeloma (MM). It is suggested that the MAGE-C1 gene in MM may be considered as an additional marker predicting the effectiveness of therapy. Purpose. Determine of MAGE-C1 gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) in MM patients and assess the correlation of expression with some laboratory parameters and response on bortezomib-containing therapy. Patients and methods. A prospective study from February 2019 to July 2021 included 76 MM patients (43 men and 33 women) aged 35 to 82 years (median 58). The level of MAGE-C1 gene expression by qPCR-PCR in bone marrow samples, enriched with CD138+ cells, was determined for all patients. Quantity of MAGE-C1 expression was calculated with 2ΔCt method relative to housekeeping gene expression. Plasma cell content ranged from 1 to 89% (median 26%), monoclonal paraprotein secretion vary from 2.1 to 82.1 g/l, Bence-Jones protein in urine (from trace values to 12.54 g/day) was determined in 50 patients. High - risk cytogenetic [t(4; 14)(p16; q32), del17p] was documented in 22 patients, bone plasmocytomas - in 36 patients, myeloma nephropathy - in 17. All patients recieved induction with bortezomib - containing courses. Median follow-up duration: 14 months (1-22 months). As a control, similar bone marrow cells of 7 healthy donors were investigated. Results. The average value of MAGE-C1 expression in donors was 0.01 ± 0.007, a range of values vary from 0.0003 to 0.06. Gene expression MAGE-C1 index > 0.06 is accepted as increased expression. High serum lactate dehydrogenase (LDH) level (higher than the upper limit of reference values) was observed in patients with high MAGE-C1 gene expression [median - 0.35], normal LDH-level (≤ 260 U/L) was observed in patients with lower MAGE-C1 gene expression [median - 0.04]; p < 0.0001 (Figure 1). Negative correlation was found between the content of hemoglobin (Hb) and level of MAGE-C1 expression: Hb ≥ 12.0 g/dl was observed in patients with increased MAGE-C1 expression [median - 0.27], and Hb 5.5-11.8 g/dl - in patients with lower expression indices [median 0.1]; p=0,034. Antitumor response was evaluated after induction therapy in 51 patients. In 100% of patients with MAGE-C1 expression of ≤ 0.06 ≥ partial response (PR) was achieved, while in patients with MAGE-C1 expression of 0.07-1.0 ≥ PR was achieved in 84.6% of cases, and in patients with MAGE-C1 expression of > 1.0 - in 58%; p > 0.05. Besides, patients with ≥ PR had a level of MAGE-C1 expression lower than patients with a refractory to bortezomib [median 0.17 vs 1.14]; p = 0.04 (Figure 2). Conclusions. High MAGE-C1 gene expression in MM patients is associated with an increase in LDH, which corresponds to stage III by R-ISS. Probability of achievement of the antitumor response ≥ PR on the bortezomib-containing induction is reliable above at patients with low MAGE-C1 expression while the hyperexpression of this gene is followed by a refractory to a bortezomib therapy. Further study of MAGE-C1 gene expression may promote a personalized approach in the choice of antitumor therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

CONTEXT:A substantial number of patients with acute lymphoblastic leukemia (ALL) die from progressive disease after relapse that is frequently associated with clonal evolution. Clinical exome sequencing (CES) is a useful tool for mutation investigation, particularly in leukemic subclones during relapse. OBJECTIVE:The objective of this pilot study was to determine the specific genetic abnormalities associated with primary tumor comparing with normal hematopoietic tissue and to define clonal evolution patterns at relapse. DESIGN:CES (TruSightTM One Sequencing Panel) was performed on samples obtained from the patient with B-cell Ph-negative ALL at diagnosis, remission and relapse. SETTING:DNA was extracted from bone marrow samples according to modified salting out method. Libraries were pooled together and underwent two rounds of hybridization and capture, and additional quantification and size assessment. Between 21 and 26 million reads for each sample were obtained with a MiSeq (Illumina, USA), with a coverage of at least 20x for 97% of all sequences. More than 99% of reads passed quality and length control and were used in the analysis. De-multiplexing of indexed reads and the generation of FASTQ files were performed by the MiSeq system on-instrument software. The sequencing data, composed of 150 bp paired-end reads, were processed for MiSeq Reporter (Illumina), which is endowed with Burrows-Wheeler Aligner (BWA) for alignment on a reference sequence of hg19 and Genome Analysis Toolkit (GATK) for variant calling. Then, the Variant Call format (VCF) file was processed for analyzing on VariantStudio (Illumina) and wANNOVAR (Yang H. et al, 2015), which contain annotation databases, such as the ClinVar database, OMIM, the dbSNP database), the Polymorphism Phenotyping v2 database (Polyphen-2), and the Sorting Intolerant from Tolerant database (SIFT) and some others. PATIENTS OR OTHER PARTICIPANTS :A 27-year-old woman, who was admitted to the National Research Center for Hematology (Moscow, Russia) on March, 2017, presented with a three-month history of paleness and ossalgia. Common B-ALL (BII) ALL with complex karyotype and extramedullary lesions in ovaries, kidneys, intra-abdominal and peripheral lymph nodes has been diagnosed. Patient achieved complete remission (CR) after first induction therapy according to ALL-2016 protocol (ClinicalTrials.gov NCT03462095). After 16 months of CR during maintenance therapy (July, 2019) was diagnosed a relapse, which was refractory to multiple chemotherapy approaches and the patient died from the disease (January, 2020). RESULTS:Primary dataset analysis included more than 9000 gene variations for each of the three samples. Only clonal variations (at diagnosis) that are pathogenic either by the type of mutations (insertions (frameshift) and splice site mutations) or by prediction databases (PolyPhen-2, SIFT) were selected. As a result, about 70 variations were identified for each of the samples. Table 1 shows all clonal somatic mutations by which tumor tissue differs from normal hematopoietic tissue. At the time of diagnosis, a clone with monosomy of chromosome 3 was identified, that disappeared at CR and reappeared as a minor clone at relapse. Taking in consideration that the loss of one allele was caused by monosomy, we searched for pathogenic variations in genes localized on the chromosome 3 in order to identify genes inactivated according to Knudson's "Two-Hit" Theory of cancer causation (Knudson A., 1971). Out of 500 genes variations on chromosome 3, mutations in 5 genes according to prediction databases (PolyPhen-2, SIFT) were defined as pathogenic. All mutations were germinal. Moreover, four out of five mutations were homozygous. Table 2 shows the dynamics of pathogenic variations in genes localized on the chromosome 3 at different stages of the disease. The missense mutation ofCOL7A1gene (c.2791C> T), which was found in a small percentage (8%), was localized on the disappeared copy of chromosome 3, and its detection in this case was possible due to the presence of normal cells. CONCLUSIONS:There is an evident change in tumor clones at relapse: a major clone diagnosed inde novoB-cell ALL patient became minor at relapse. The data obtained from this analysis suggest that at the time of diagnosis, several clones are present in the tumor, and that most of the diagnosed pathogenic variations are not associated with oncogenesis and are germinal. Disclosures No relevant conflicts of interest to declare.

Context Testing for NRAS, KRAS, JAK2, CRLF2 and TP53 mutations in adult ALL patients and studying of molecular pathways are critical for understanding the biology of ALL. Objective Assessment of mutations in NRAS, KRAS, JAK2, CRLF2, TP53 genes and analysis of signaling pathways in de novo adult ALL patients. Design NRAS, KRAS, JAK2, CRLF2, TP53 mutations were studied B-ALL (Ph+/Ph−). TP53 gene mutations were investigated in B-ALL (Ph+/Ph−) and T-ALL patients.TP53 gene was also evaluated in non-tumor cells of 5 patients to exclude germline mutations. Gene expression profiling was performed for 5 B-ALL patients. Setting Sanger sequencing of hot-spot regions NRAS, KRAS, JAK2, CRLF2 and TP53 (exons 2-10) was conducted. PanCancer Pathways Panel by NanoString was used for gene expression analysis. KEGG pathway analysis for overexpressed genes (of the 13 canonical pathways) was carried out using the DAVID tool. Patients or other participants The study included 95 B-ALL patients, m:f (39:45), median age 31 years; 64 T-ALL, m:f (44:20), median age 27 years, 27 Ph+ ALL, m:f (10:17), median age 33 years. 109 patients treated by protocol ALL-2016 ( NCT03462095 ), 50 patients – by ALL-2009 ( NCT01193933 ), 23 – by ALL-2012 (for Ph«+»ALL) and 4 – by other protocols. Median follow-up 20.8 months. Results NRAS (9.7%), KRAS (12.9%), JAK2 (3.16%) mutations was detected only in B-ALL patients. CRLF2 mutations mutation was not detected. NRAS, KRAS, JAK2 mutations didn't impact disease outcome in B-ALL patients treated by ALL-2009, ALL-2016 protocols. TP53 mutations more often were detected in B-ALL patients (11.2%) compared with T (3.1%) and Ph+ (7.4%). They were associated with poorer OS (TP53-WT – 66.9% vs 48% TP53 mut, p=0.094) and a lower incidence of CR (TP53-WT – 90.6% vs 70.4% TP53 mut, p = 0.033). In 4 out of 5 patients, TP53 mutations were detected in non-tumor cells what considers leukemia as a manifestation of Li-Fraumeni syndrome. The most significant pathways affected were pathways in cancer (p = 6.7E-33) and cell cycle (p = 3.3E-31). Conclusions TP53 mutations are associated with unfavorable prognosis. Most TP53 mutations are of hereditary origin. NRAS, KRAS, JAK2 mutations did not affect the disease outcome. Pathway analysis provides a useful tool for understanding the ALL biology, and we plan to continue it and recruit more patients. The reported study was funded by RFBR, project number 19-31-27001.

Clonal composition of human multipotent mesenchymal stromal cells (MMSCs) labeled with lentiviral vectors carrying genetic barcodes was studied. MMSCs were transduced with a cloned library of self-inactivating lentiviral vectors carrying 667 unique barcodes. At each cell culture passage, 120 cells were plated one cell per well in 96-well plates. The efficiency of cloning and labeling of the clonogenic cells was determined. DNA was extracted from the cell-derived colonies, and the barcodes were identified by Sanger sequencing. Also, DNA was extracted from the total MMSC population at each passage to analyze the diversity and representation of barcodes by deep sequencing using the Illumina platform. It was shown that the portion of MMSCs labeled with the lentiviral vectors remained stable in the passaged cells. Because of the high multiplicity of infection, the labeling procedure could decrease the proliferative potential of MMSCs. Identification of barcodes in individual cell clones confirmed the polyclonal character of the MMSC population. Clonal composition of MMSCs changed significantly with the passages due to the depletion of proliferative potential of most cells. Large clones were found at the first passage; at later passages, many small clones with a limited proliferative potential were detected in the population. The results of deep sequencing confirmed changes in the clonal composition of MMSCs. The polyclonal MMSC population contained only a small number of cells with a high proliferative potential, some of which could be stem cells. MMSCs with a high proliferative potential were detected more often in the earliest passages. In this regard, we would recommend to use MMSCs of early passages for regenerative medicine applications based on cell proliferation.

Comprehensive analysis of molecular pathology requires a collection of reference samples representing normal tissues from healthy donors. For the available limited collections of normal tissues from postmortal donors, there is a problem of data incompatibility, as different datasets generated using different experimental platforms often cannot be merged in a single panel. Here, we constructed and deposited the gene expression database of normal human tissues based on uniformly screened original sequencing data. In total, 142 solid tissue samples representing 20 organs were taken from post-mortal human healthy donors of different age killed in road accidents no later than 36 hours after death. Blood samples were taken from 17 healthy volunteers. We then compared them with the 758 transcriptomic profiles taken from the other databases. We found that overall 463 biosamples showed tissue-specific rather than platform- or database-specific clustering and could be aggregated in a single database termed Oncobox Atlas of Normal Tissue Expression (ANTE). Our data will be useful to all those working with the analysis of human gene expression., Design Type(s)gene expression analysis objective • data integration objective • organism part comparison design • transcription profiling designMeasurement Type(s)transcription profiling assayTechnology Type(s)RNA sequencingFactor Type(s)sex • age • organism subdivisionSample Characteristic(s)Homo sapiens • kidney • Kidney • Colon • Liver • brain • Lung • endometrium • Ovary • prostate gland • Esophagus • Stomach • Mammary gland • Thyroid gland • Pancreas • Tonsil • skeletal muscle tissue • small intestine • Adrenal gland • urinary bladder • skin of body • uterus • uterine cervix • Small intestine • tonsil • blood • bone marrow • esophagus • liver Machine-accessible metadata file describing the reported data (ISA-Tab format)

Hemophilia B is a hereditary X-linked coagulation disorder. This pathology is caused by various defects in the factor IX gene, which is, being about 34 kb long and consisting of eight exons, localized in the Xq27 locus of the X-chromosome long arm. Mutations were revealed in 56 unrelated patients with hemophilia B in this study by using direct sequencing of factor IX gene functionally important fragments. Forty-six mutations were found with prevailing missense mutations (n = 30). The rest of the mutations were nonsense (n = 4) and splicing (n = 4) mutations, large deletions (n = 3), microdeletions (n = 2), microinsertions (n = 2), and promoter mutations (n = 1). Eleven of 46 mutations were previously unknown for human populations.

Acute intermittent porphyria (AIP) is the most common and severe form of porphyrias. This is a dominant inherited disorder with low penetrance, caused by mutations in gene coding hydroxymethylbilane synthase (HMBS). We present the results of our long-term genetic study of AIP patients and their relatives (N = 153 and 302, respectively). We detected 88 HMBS gene mutations, 24 of which never described before. To identify additional factors conditioning AIP manifestation, we carried out whole exome sequencing on the group of AIP patients (N = 6). Mutation spectra of different patients virtually did not overlap. In 5 out of 6 patients, we found defects in genes regulating nervous system (UNC13A, ALG8, FBXO38, AGRN, DOK7, SCN4A). As usually acute AIP attacks have various neurological symptoms, we proposed a hypothesis of possible contribution of mutations in such genes in AIP manifestation.

In contrast to the abundant fossil record of arctic ground squirrels, Urocitellus parryii, from eastern Beringia, only a limited number of fossils is known from its western part. In 1946, unnamed GULAG prisoners discovered a nest with three mummified carcasses of arctic ground squirrels in the permafrost sediments of the El’ga river, Yakutia, Russia, that were later attributed to a new species, Citellus (Urocitellus) glacialis Vinogr. To verify this assignment and to explore phylogenetic relationships between ancient and present-day arctic ground squirrels, we performed 14C dating and ancient DNA analyses of one of the El’ga mummies and four contemporaneous fossils from Duvanny Yar, northeastern Yakutia. Phylogenetic reconstructions, based on complete cytochrome b gene sequences of five Late Pleistocene arctic ground squirrels and those of modern U. parryii from 21 locations across western Beringia, provided no support for earlier proposals that ancient arctic ground squirrels from Siberia constitute a distinct species. In fact, we observed genetic continuity of the glacialis mitochondrial DNA lineage in modern U. parryii of the Kamchatka peninsula. When viewed in a broader geographic perspective, our findings provide new insights into the genetic history of U. parryii in Late Pleistocene Beringia.

Hemophilia B is a hereditary X-linked coagulation disorder. This pathology is caused by various defects in the factor IX gene, which is, being about 34 kb long and consisting of eight exons, localized in the Xq27 locus of the. X-chromosome long arm. Mutations were revealed in 56 unrelated patients with hemophilia B in this study by using direct sequencing of factor IX gene functionally important fragments. Forty-six mutations were found with prevailing missense mutations (n = 30). The rest of the mutations were nonsense (n = 4) and splicing (n = 4) mutations, large deletions (n = 3), microdeletions (n = 2), microinsertions (n = 2), and promoter mutations (n = 1). Eleven of 46 mutations were previously unknown for human populations.

The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic "barcodes" has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.

Acute intermittent porphyria (AIP) is an autosomal dominant hereditary disease, caused by partial deficiency of porphobilinogen deaminase (PBGD), one of the key enzymes ofheme biosynthesis. This study describes molecular genetics of AIP in Russia. Mutation analysis of PBGD gene in 70 unrelated patients revealed 47 various genetic defects, 28 of which had not been described previously. Mutations 53delT and Argl 73 Trp (recorded 8 times, in total 23%) proved to be the most common in Russia. Microdeletion 53delThas monophyletic origin and was found only in Russia. Molecular genetic examination of 132 relatives of AIP patients from 40 families revealed 52 latent carriers of the disease. Low (about 10%) AIP penetrance indicates that a mutation in the PBGD gene is an important but not sufficient prerequisite for clinical manifestation of the disease. Modulation of penetrance in erythropoietic protoporphyria by coinheritance of a mutant allele and a functionally defective wild type allele of ferrochetalase gene has been shown previously. We hypothesized that similar mechanism works in AIP. Sequencing of the full length PBGD genes from unrelated AIP patients as well as SN P analysis, and the analysis of abnormal PBGD mRNA splicing showed that in case ofAIP, this hypothesis is not true and some other factors are responsible for the penetrance of this disease.

The ranges of the great tit Parus major and the Japanese tit P. minor overlap in the middle Amur region, where hybridization of these two species occur. These species have contacted for nearly a century on the western slope of the Malyi Khingan Ridge (the central part of the sympatry zone), but the great tit has colonized territories to the east of the ridge only in the last two decades. The percentage of the P. minor’s allele of intron 2 of the mioglobin gene has significantly increased from 8.9% in the west to 27.8% in the east in phenotypically major’s populations. Thus, the percentage of foreign mtDNA in P. major populations did not change significantly from west (6.2%, n = 120) to east (3.2%, n = 61). Simultaneous use of two genetic markers (one nuclear and the other mitochondrial) supports our conclusion on strong introgression in the populations of both species, which nevertheless maintain their morphological specificity in the contact zone.

In search of new efficient markers for genetic diagnostics of hemophilia A, two tri-nucleotide microsatellite repeats (STR) at chromosome X loci, which flank coagulation factor VIII gene (F8), namely STR HA472—CTT-repeat, which is localized adjacent to the GAB3 gene 163 kb apart from the 3′ end of the F8 gene and STR HA544—repeat (CTT) x (ATT) y located at a distance of 375 kb from the 5′ end of the F8 gene were discovered. Detailed analysis using PCR and sequencing has shown that STR HA472 contains two long variable CTT-blocks separated by small spacer CCTCCC. The location of recognition site of restriction endonuclease MnlI (CCTC) in the spacer permits to test differentially the polymorphic blocks and thus to increase the analysis informativity. STR HA544 is also represented by two polymorphic blocks (CTT and ATT), for separate amplification of which highly informative PCR amplification assays were elaborated. The study has been done using DNA samples of 212 individuals (125 women) from 48 families with hemophilia A carriers. Our results point to Mendelian inheritance of the markers studied, a high number of allelic variants and high heterozygosity, which was 90 and 100% for HA544 and HA472, respectively. This permitted us to use these data for practical gene diagnostics of the carriers and prenatal diagnostics of hemophilia A. In addition to high informativity STR HA472 and HA544 are highly important for diagnostics as they are located at a shorter distance than other known extragenic polymorphisms of the F8 gene. In contrast to dinucleotide repeats, trinucleotide repeats are readily tested, not requiring high-resolution electrophoretic systems. In addition, they are located on the opposite sites of the F8 gene. This permits to control homologous recombination events in the locus and thus to prevent diagnostic mistakes.

Frequencies of the C/T SNP alleles at position 2403 of the human coagulation factor VIII gene intron 1, containing the AluI restriction endonuclease recognition site, were examined. Genomic DNA samples for the analysis were obtained from the consulted women and their relatives from the families with hemophilia A. A total of 221 unrelated X chromosomes were studied. The two allelic variants were found with similar frequencies of T(Alu+), 0.53 and C(Alu−), 0.47. The heterozygosity index evaluated as equal to 0.50 was correlated with the experimental heterozygote number. The absence of a tight linkage between the AluI SNP and the widely used in the hemophilia A gene diagnostics HindIII polymorphism (C/T SNP at position 103 of intron 19) was demonstrated. Summarized informativity of these two markers for obligate carriers and for those detected in this study constituted 68% (32 out of 47). At the same time using one of the markers, only 40% (HindIII) and 51% (AluI) of the consulted women were informative. The new marker was used in 13 prenatal DNA diagnostics of hemophilia A. A new deletion polymorphism (del TGA, position 2281–2283 of intron 1) was described in close proximity of the AluI SNP with the frequency of about 0.05. among the five other SNP of the factor VIII gene examined (Bme18I, intron 1; HpaII, intron 13; MnlI, exon 14; Bst4CI, exon 25; and MseI, exon 26) no effective diagnostic markers were found. Only the MnlI polymorphism could be recommended for limited usage.

In four ground squirrel species from the Volga region—yellow (Spermophilus fulvus), russet (S. major), little (S. pygmaeus), and speckled (S. suslicus)—four hybridization variants (major/fulvus, major/pygmaeus, major/suslicus, and pygmaeus/suslicus) have been reliably described. Earlier we have shown that populations of S. major from the Volga region were characterized by wide introgression of mtDNA from S. fulvus and S. pygmaeus, which probably, resulted from ancient hybridization [5]. In this study, the same populations were used to analyze the introgression of the Y chromosome, which (unlike mtDNA) is paternally inherited. Three genes, ZfY, SRY, and SmcY were tested as Y-chromosomal candidate markers. It was demonstrated that Y chromosome of ground squirrels lacked the ZfY gene, while its homologous structure, ZfY(X), was presumably linked to the X chromosome. The SRY region examined was rather conservative. In particular, the sequences determined in S. major and S. fulvus were identical, while three out of four substitutions found in S. pygmaeus were located in the coding region. The SmcY gene was found to be the most suitable marker, providing distinguishing of all of the four ground squirrel species by nine nucleotide substitutions. Introgression at the Y chromosome was observed only in two cases: in one S. major individual (out of 51 phenotypically pure animals) caught in the major/fulvus sympatry zone, and in four (one litter) out of fourteen S. fulvus individuals caught in close vicinity of the sympatry zone of these two species. Among 28 S. pymaeus and 9 S. suslicus individuals, no foreign SmcY genes were detected. Two colonies of the “hybrid swarm” type were examined with eight major/suslicus hybrids analyzed in the first and seventeen major/fulvus hybrids in the second colony. The prevalence of the S. major paternal lineages was observed in both colonies (87.5 and 82.4%, respectively). The data obtained suggest that compared to wide mtDNA introgression, introgression of Y chromosome in the Volga region ground squirrels is statistically significantly less frequent event.

The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’.

Four species of ground squirrel—yellow (Spermophilus fulvus), russet (S. major), small (S. pygmaeus), and spotted (S. suslicus)—occur in the Volga region. Between S. major and S. pigmaeus, S. major and S. fulvus, and S. major and S. suslicus, sporadic hybridization was reported. Using sequencing and restriction analysis, we have examined the mtDNA C region in 13 yellow, 60 russet, 61 small, 45 spotted ground squirrels, and 9 phenotypic hybrids between these species. It was shown that 43% of S. major individuals had “alien” mitotypes typical of S. fulvus and S. pygmaeus. Alien mitotypes occurred both within and outside sympatric zones. No alien mitotypes were found in 119 animals of the other three species, which suggests that only one parental species (S. major) predominantly participates in backcrosses. Phenotypic hybrids S. fulvus × S. major and S. major × S. pygmaeus) were reliably identified using RAPD–PCR of nuclear DNA. However, we could find no significant traces of hybridization in S. major with alien mitotypes. Analysis of p53 pseudogenes of S. major and S. fulvus that were for the first time described in the present study produced similar results: 59 out of 60 individuals of S. major (including S. major with S. fulvus mitotypes) had only the pseudogene variant specific for S. major. This situation is possible even at low hybridization frequencies (less than 1% according to field observations and 1.4 to 2.7% according to nuclear DNA analysis) if dispersal of S. major from the sympatric zones mainly involved animals that obtained alien mtDNA via backcrossing. The prevalence of animals with alien mitotypes in some S. major populations can be explained by the founder effect. Further studies based on large samples are required for clarifying the discrepancies between mitochondrial and nuclear DNA data.

Porphobilinogen deaminase (PBGD) is a key enzyme of the heme biosynthetic pathway. Defects in the PBGD gene lead to an autosomal dominant disease, acute intermittent porphyria (AIP). Almost all AIP patients with rare exceptions are heterozygous for the defective gene. To date, at least 160 different mutations causing AIP are identified. Extensive investigations along this line are conducted in many countries of the world. In Russia these studies had not been hitherto performed. Here we report the results of molecular genetic examination of four Russian patients with AIP diagnosed from clinical symptoms. By direct sequencing of the PBGD gene or the corresponding cDNA, we have detected four mutations, three of which were not previously encountered in the world population. These are TAAG deletion in intron 7 between positions +2 and +5 (IVS7 2–5 delTAAG); T deletion in the initiation codon ATG of exon 3, and the G for C replacement at position –1 of intron 5 (IVS5 as –1 G–C), which disrupts splicing. In addition, in one female patient, a known deletion CT in codon 68 was revealed. In two patients, expression of PBGD gene alleles was significantly disproportional, so that normal mRNA prevailed in one case and mutant mRNA of nonerythroid type in the other. Deletion in intron 7 was easily detectable due to the formation of a heteroduplex fragment with abnormal electrophoretic mobility directly in PCR. This simple heteroduplex analysis allowed us to exclude AIP carriage in son and daughter of a female patient with the genetic defect.

The ranges of the great tit Parus major and the Japanese tit P. minor overlap in the middle Amur region, where hybridization of these two species occur. These species have contacted for nearly a century on the western slope of the Malyi Khingan Ridge (the central part of the sympatry zone), but the great tit has colonized territories to the east of the ridge only in the last two decades. The percentage of the P. minor's allele of intron 2 of the mioglobin gene has significantly increased from 8.9% in the west to 27.8% in the east in phenotypically major's populations. Thus, the percentage of foreign mtDNA in P. major populations did not change significantly from west (6.2%, n = 120) to east (3.2%, n = 61). Simultaneous use of two genetic markers (one nuclear and the other mitochondrial) supports our conclusion on strong introgression in the populations of both species, which nevertheless maintain their morphological specificity in the contact zone.

We developed a method for gene transfer into mesenchymal stromal cells. Lentivirus vector containing green fluorescent protein gene for labeling stromal and hemopoietic precursor cells was obtained using two plasmid sets from different sources. The vector was injected into the femur of mice in vivo and added into culture medium for in vitro infection of the stromal sublayer of long-term bone marrow culture. From 25 to 80% hemopoietic stem cells forming colonies in the spleen were infected with lentivirus vector in vivo and in vitro. Fibroblast colony-forming cells from the femoral bones of mice injected with the lentivirus vector carried no marker gene. The marker gene was detected in differentiated descendants from mesenchymal stem cells (bone cavity cells from the focus of ectopic hemopoiesis formed after implantation of the femoral bone marrow cylinder infected with lentivirus vector under the renal capsule of syngeneic recipient). In in vitro experiments, the marker gene was detected in sublayers of long-term bone marrow cultures infected after preliminary 28-week culturing, when hemopoiesis was completely exhausted. The efficiency of infection of stromal precursor cells depended on the source of lentivirus. The possibility of transfering the target gene into hemopoietic precursor cells in vivo is demonstrated. Stromal precursor cells can incorporate the provirus in vivo and in vitro, but conditions and infection system for effective infection should be thoroughly selected.

to estimate the spread of weak D antigen types of the Rhesus system in the citizens of the Russian Federation and a possibility of serologically identifying these types.The red blood cells and DNA of people with weakened expression of D antigen were investigated using erythrocyte agglutination reaction in salt medium (2 methods); agglutination reaction in the gel columns containing IgM + IgG anti-D antibodies, indirect antiglobulin test with IgG anti-D antibodies (2 methods); polymerase chain reaction to establish the type of weak D.A rhesus phenotype was determined in 5100 people in 2014-2015. The weakened agglutinable properties of red blood cells were detected in 102 (2%) examinees. 63 examinees underwent genotyping to identify the variants of the weak D antigen, which identified 6 weak D types. There were the most common weak D types 3 (n=31 (49.2%)) and weak D type 1 (n=18 (28.6%)), including weak D type 1.1 in one (1.6%) case. The other 4 weak D antigen types were as follows: weak D type 2 (14.3% (n=9)), weak D type 15 (4.8% (n=3)), weak D type 4.2 (DAR) (1.6% (n=1)) and weak D type 6 (1.6% (n=1)). The antiglobulin test in the gel column containing antiglobulin serum was the most sensitive serological assay to identify the weak D antigen. Only a molecular test could establish weak D type 15 in 2 samples of red blood cells with Ccdee and ccdEe phenotypes.The weak D antigen could be serologically identified in 96.8% of cases. When testing for weak D, particular attention should be given to people with the D-negative phenotype who had the C or E antigens. Our investigations conducted for the first time in Russia will be able to improve the immunological safety of red blood cell-containing medium transfusions for patients.Цель исследования. Оценка распространенности типов слабого антигена D (weak D) системы резус у россиян и возможности выявления этих типов серологическими методами. Материалы и методы. Исследовали эритроциты и ДНК людей с ослабленной экспрессией антигена D с помощью реакции агглютинации эритроцитов в солевой среде (2 метода); реакции агглютинации в гелевых колонках с IgM+IgG анти-D-антителами, непрямого антиглобулинового теста с IgG анти-D-антителами (2 метода); полимеразной цепной реакции для установления типа weak D. Результаты. В 2014-2015 гг. резус-фенотип определен у 5100 человек. Ослабление агглютинабельных свойств эритроцитов выявлено у 102 (2%) обследованных. Генотипирование для идентификации вариантов антигена weak D проведено у 63 обследованных, выявлено 6 типов weak D. Наиболее частыми оказались типы weak D type 3 (n=31; или 49,2%) и weak D type 1 (n=18; или 28,6%), в том числе в одном случае weak D type 1.1 (1,6%). Остальные 4 типа антигена weak D имели следующие частоты: weak D type 2 - 14,3% (n=9), weak D type 15 - 4,8% (n=3), weak D type 4.2 (DAR) (n=1) и weak D type 6 (n=1) - по 1,6% каждый. Наиболее чувствительным серологическим методом идентификации антигена weak D был антиглобулиновый тест в гелевых колонках, содержащих антиглобулиновую сыворотку. В 2 образцах эритроцитов с фенотипами Ccdee и ccdEe наличие weak D type 15 установлено только молекулярным методом. Заключение. Серологическими методами слабый антиген D выявлен в 96,8% случаев. Особое внимание при проверке на наличие weak D следует уделять людям с D-отрицательным фенотипом, имеющим антигены С или Е. Наши исследования, проведенные впервые в России, позволят улучшить иммунологическое обеспечение безопасности трансфузий пациентам сред, содержащих эритроциты.

In search of new efficient markers for genetic diagnostics of hemophilia A, two tri-nucleotide microsatellite repeats (STR) at chromosome X loci, which flank coagulation factor VIII gene (F8), namely STR HA472--CTT-repeat, which is localized adjacent to the GAB3 gene 163 bp apart from the 3' end of the F8 gene and STR HA544--repeat (CTT)x(ATT)y located at a distance of 375 bp from the 5' end of the F8 gene were discovered. Detailed analysis using PCR and sequencing has shown that STR HA472 contains two long variable CTT-blocks separated by small spacer CCTCCC. The location of recognition site of restriction endonuclease Mnl1 (CCTC) in the spacer permits to test differentially the polymorphic blocks and thus to increase the analysis informativity. STR HA544 is also represented by two polymorphic blocks (CTT and ATT), for separate amplification of which highly informative PCR amplification assays were elaborated. The study has been done using DNA samples of 212 individuals (125 women) from 48 families with hemophilia A carriers. Our results point to Mendelian inheritance of the markers studied, a high number of allelic variants and high heterozygosity, which was 90% and 100% for HA544 and HA472, respectively. This permitted us to use these data for practical gene diagnostics of the carriers and prenatal diagnostics of hemophilia A. In addition to high informativity STR HA472 and HA544 are highly important for diagnostics as they are located at a shorter distance than other known extragenic polymorphisms of the F8 gene. In contrast to dinucleotide repeats, trinucleotide repeats are readily tested, not requiring high-resolution electrophoretic systems. In addition, they are located on the opposite sites of the F8 gene. This permits to control homologous recombination events in the locus and thus to prevent diagnostic mistakes.

Frequencies of the CIT SNP alleles at position 2403 of the human coagulation factor VIII gene intron 1, containing the AluI restriction endonuclease recognition site, were examined. Genomic DNA samples for the analysis were obtained from the consulted women and their relatives from the families with hemophilia A. A total of 221 unrelated X chromosomes were studied. The two allelic variants were found with similar frequencies of T(Alu+), 0.53 and C(Alu-), 0.47. The heterozygosity index evaluated as equal to 0.50 was correlated with the experimental heterozygote number. The absence of a tight linkage between the AluI SNP and the widely used in the hemophilia A gene diagnostics HindIII polymorphism (CIT SNP at position 103 of intron 19) was demonstrated. Summarized informativity of these two markers for obligate carriers and for those detected in this study constituted 68% (32 out of 47). At the same time using one of the markers, only 40% (HindIII) and 51% (AluI) of the consulted women were informative. The new marker was used in 13 prenatal DNA diagnostics of hemophilia A. A new deletion polymorphism (del TGA, position 2281-2283 of intron 1) was described in close proximity of the AluI SNP with the frequency of about 0.05. among the five other SNP of the factor VIII gene examined (Bme 18I, intron 1; HpaII, intron 13; MnlI, exon 14; Bst4CI, exon 25; and MseI, exon 26) no effective diagnostic markers were found. Only the MnlI polymorphism could be recommended for limited usage.

Introduction. Patients (pts) with oncopathology who have hyperexpression of c-MYC demonstrate low response to chemotherapy and poor prognosis. In our study we investigated the level of c-MYC gene expression in pts withmultiple myeloma (MM) and/or monoclonal gammopathy undetermined significance (MGUS) at the moment of MM/MGUS diagnosis. Patients and Methods. 40 patients with newly diagnosed MM/MGUS and 9 healthy bone marrow donors (as a control group) were included in our study. The median age of patients with MM/MGUS was 56 years (29-77), donors - 32 years (24-41). The bone marrow collection for examination was done at the moment of diagnosis. The investigated material was divided into 2 samples: all mononuclears and separated CD138+ cells. Level of c-MYC expression was identified by quantitative real time PCR (qRT-PCR). The whole number of qRT-PCR analysis was 98. Induction therapy (VCD/PAD) was initiated for 36 patients with MM, 4 patients with MGUS remained under observation. Antitumor response was evaluated after induction therapy according to the IMGG criteria. Results. From 98 samples among pts reliable results qRT-PCR and evaluated c-MYC expression were in 36 samples from total mononuclears and 17 from separated CD138+ cells. In all 18 samples from healthy donors: 9 from total mononuclear, and 9 from CD138+ cells results of qRT-PCR were reliable. Mean c-MYC expression level among MM/MGUS pts was 55.014 (2ΔCt) in CD138+ cells and in samples from total mononuclears - 17.585 (2ΔCt). In healthy donors the results were 7.183(2ΔCt) and 1.907 (2ΔCt) respectively. After induction therapy complete response (CR) was demonstrated 6 patients, very good partial response (VGPR) - in 7 patients, partial response (PR) - in 15 and other 5 pts were refractory. Mean c-MYC expression level in samples from separated CD138+ cells in pts with CR was-14.259 (2ΔCt), with VGPR-38.314 (2ΔCt), with PR-50.506 (2ΔCt), and in refractory pts-181.271 (2ΔCt), among patients with MGUS the level was 10.928 (2ΔCt). In samples from mononuclears: 5.138, 30.355, 11.877, 38.911, 7.323 (2ΔCt) respectively. Thus, response after induction therapy was worse among patients with high gene c-MYC expression (Figure 1 a). Such a correlation was not revealed in analysis of data from samples of total mononuclears (Figure 1 b). Conclusion. Mean c-MYC gene expression level in CD138+ cells among MM/MGUS pts was higher than among healthy donors (p Disclosures No relevant conflicts of interest to declare.

In four ground squirrel species from the Volga region-yellow (Spermophilus fulvus), russet (S. major), little (S. pygmaeus), and speckled (S. suslicus)--four hybridization variants (major/fulvus, major/pygmaeus, major/suslicus, and pygmaeus/suslicus) have been reliably described. Earlier we have shown that populations of S. major from the Volga region were characterized by wide introgression of mtDNA from S. fulvus and S. pygmaeus, which probably, resulted from ancient hybridization. In this study, the same populations were used to analyze the introgression of the Y chromosome, which (unlike mtDNA) is paternally inherited. Three genes, ZfY, SRY, and SmcY were tested as Y-chromosomal candidate markers. It was demonstrated that Y chromosome of ground squirrels lacked the ZfY gene, while its homologous structure, ZfY(X), was presumably linked to the X chromosome. The SRY region examined was rather conservative. In particular, the sequences determined in S. major and S. fulvus were identical, while three out of four substitutions found in S. pygmaeus were located in the coding region. The SmcY gene was found to be the most suitable marker, providing distinguishing of all of the four ground squirrel species by nine nucleotide substitutions. Introgression at the Y chromosome was observed only in two cases: in one S. major individual (out of 51 phenotypically pure animals) caught in the major/fulvus sympatry zone, and in four (one litter) out of fourteen S. fulvus individuals caught in close vicinity of the sympatry zone of these two species. Among 28 S. pymaeus and 9 S. suslicus individuals, no foreign SmcY genes were detected. Two colonies of the "hybrid accumulation" type were examined with eight major/suslicus hybrids analyzed in the first and seventeen major/fulvus hybrids in the second colony. The prevalence of the S. major paternal lineages was observed in both colonies (87.5 and 82.4%, respectively). The data obtained suggest that compared to wide mtDNA introgression, introgression of Y chromosome in the Volga region ground squirrels is statistically significantly less frequent event.

To investigate effectiveness of allogenic transplantation of the bone marrow (TBM) in the treatment of hemoblastosis patients from a high risk group, the course of donor bone marrow retention, tolerance and antitumor activity of this therapy.11 patients received TBM in low-intensity regimen in Hematological Research Center in 1999-2001. All the patients were from a high risk group. Conditioning was based on the combination of fludarabin with busulfan. The transplanted precursor cells were taken from the bone marrow and/or peripheral donor blood. The retention was controlled by differential agglutination of erythrocytes and amplification of hypervariable sites of DNA. Minimal residual disease was controlled by standard cytogenetical tests, fluorescent in situ hybridization or reverse-transcriptase polymerase chain reaction.All the patients tolerated pretransplantation conditioning well. By chimerism, signs of retention of donor bone marrow on day +30 after TBM were observed in 9 patients of 11. Acute graft versus host reaction developed in 5 patients. This reaction was treated conventionally with methylprednisone and cyclosporin A, in 4 cases with a good effect. A complete remission persists in 5 patients. Mean follow-up lasted for 241 days.Thus, transplantation was successful in 50% patients with an unfavourable prognosis who are still in a complete remission. This suggests efficacy of the above method of treatment.