GTG Banding Research Articles

70. PREIMPLANTATION GENETIC TESTING FOR YQH-AUTOSOME TRANSLOCATION CARRIERS: ANALYSIS OF GAMETE SEGREGATION

Introduction Y-autosome translocations occur in approximately 1:2000 in population and represent special group of reciprocal exchanges. Due to a balanced nature they do not influence on phenotype in general. However, Y-autosome translocation can dislocate sex bivalent formation and chromosome synapsis in the first meiotic division prophase that led to gamete aneuploidy or pachytene arrest. More often Y-derived DNA, predominantly Yq12 band, is translocated onto the short arm of acrocentric chromosomes, mainly chromosomes 15 and 22 due to a strong correlation in their satellite DNA sequences. In women inheritance of such derivative acrocentric chromosomes could be associated with the risk of ovarian malignancy, but the particular mechanism of this pathological effect is not entirely clear since the Yq12 band consists of tandemly organized satellite DNA sequences. It can be associated with the transcription of satellite DNAwhich occurs during carcinogenesis. Material & methods Two families with Yq12/15p and one – with Yq12/22p translocations were analyzed during IVF cycle with PGT in International Centre for Reproductive Medicine. The mean age of men and women was 36±2.8 and 34.25±1.4 years respectively. Karyotyping analysis was performed using GTG banding technique on metaphase chromosomes from peripheral blood lymphocytes. Fluorescence in situ hybridization (FISH) was used to clarify the breakpoint on chromosomes and confirm the chromosomes-specificity of the probes. Additionally, seven chromosomes (13, 14, 15 or 22, 16, 18, 21, X), which are not involved in rearrangement, were analyzed. Results FISH with chromosome specific probes was performed on blastomere from cleavage-stage embryo. A total of 42 blastomeres were analyzed. In 43% of the blastomeres «adjacent-1» segregationwith derivate autosome and X or Y chromosomes was revealed. Segregation 3:1 was observed in 10% of the cells, with no predominant inheritance of the derivative autosome. In 21% of cells the type of chromosome segregation could not be determined due to mosaicismor polyploidy. 45% of the cells were aneuploid for autosomes, which are not involved in the translocation, which may be connected with interchromosomal effect. Conclusions Carriers of Yq12/15p or Yq12/22p translocations have a predominant inheritance of the derivative autosome, which can be associated with the disruption ofsex bivalent segregation. This research was supported by Russian Foundation for Basic Research (grant # 18-34-00279 ).

Cytogenetic anomalies in Multiple Myeloma patients:A single center study

Conventional karyotyping in the patients with Multiple myeloma (MM) is very important. Because chromosomal abnormalities which detected in these patients have diagnostic and prognostic value. In this retrospective study we aim to evaluate cytogenetic abnormalities in 133 MM patients which diagnosed at the Hematology Department of Hacettepe University. Samples were treated with trypsin and stained with Giemsa (GTG banding). 20 metaphases of each patient were examined and karyotypes were formed. Cytogenetic results of the patient’s bone marrow samples were not obtained in 19 patients, while in 116 patients karyotyping was performed. Among of these 116 patients showed that 80 patients had normal karyotpe while 34 patients had abnormal karyotypes. Both numerical and structural chromosomal anomalies were detected in patients with abnormal karyotype. Numerical and structural anomalies of chromosomes 1, 9, 16 and 13 were detected most frequently among these complex karyotypes. The anomalies we found in our patient group were consistent with the literature.

Specific Chromosomal Aberrations in Primary Amenorrhoea: Study on 3776 Cases from Indian Population.

To verify the prevalence of chromosomal abnormalities in women with primary amenorrhoea in India aiming at appropriate genetic counselling. In a 16-year retrospective (2001-2016) study, 3776 women with primary amenorrhoea were evaluated. Chromosomal analysis of all the cases was done by GTG banding. Clinical history and other laboratory findings were taken into consideration to determine the diagnosis. The karyotype results revealed 31.2% cases with chromosomal abnormalities (n = 1177/3776). In patients with abnormal chromosome complement, 31.2% exhibited numerical aberrations (n = 367) and 34.9% with structural aberrations (n = 411). About 33.9% of cases were with XY male karyotype (n = 399). As per the literature till date, this study is the largest with high incidence of chromosomal abnormalities; early detection of abnormalities is necessary for guidance to reproductive management and genetic counselling.

Neonate with 10q Interstitial Deletion within the Long Arm of Chromosome 10- A Case Report and Literature Review

Introduction: Partial deletion of distal chromosome 10q was first reported in 1978 by Lewandowski1. Interstitial deletions within bands 10q25e10q26.3 are rare. Seven such cases were reported so far2. Patient Information: A term AGA male newborn was delivered at our perinatal center with antenatal diagnosis of unbalanced translocation of chromosomes 10 and 12, and fetal cleft lip and cleft palate. Blood was sent for chromosome analysis using GTG banding method. Baby had facial dysmorphism, left cleft lip, bilateral cleft of soft and hard palate, intact nasal septum, normal ears and micrognathus. Abdominal ultrasound showed absence of right testis in inguinal canal and abdomen (anorchia). Hospital course was unremarkable except for feeding problems requiring feeding team, plastic surgery planned at 2- 3 months of age, and taping of the cleft lip. He went home on day 7. Conclusion: Here, we reported an extremely rare case of a male newborn with an interstitial deletion within the long arm of chromosome 10 between bands 10q25.1 and 10q26.1, with dysmorphic features, along with a few unreported associations (atypical Pierre-Robin sequence and bilateral dorsal horn ventriculomegaly). We added a comprehensive review of literature on chromosome 10q deletions to the case report. We also listed clinical implications of 71 RefSeq and OMIM genes noted in that ~13.3Mb 10q deletion in the Appendix. Earlier detection of both common and rare chromosomal- genetic abnormalities might prepare the family and health care team to plan optimal care to the mother, and baby.

A De Novo Deletion of Chromosome 18p with Persistent Limb Tremor and Difficulty Speaking

Background: The common causes of 18p deletion syndrome are spontaneous errors in the chromosomal structure in the early stages of human embryonic development. Clinical Presentation and Intervention: In this study, a 29-year-old girl was introduced with the features of deletion of chromosome 18. In addition, GTG banding karyotype revealed that this case had a deletion involving the short arm of chromosome 18. In comparison with the usual phenotype of 18p deletion, many phenotypical features of this case were similar to the other cases of 18p monosomy. Conclusion: However, two new features; difficulty in speaking and persistent limb tremor, were found that had not been observed in previous studies on the 18p deletion. Speaking was without obvious pronunciation, and the patient’s physical movements were always unbalanced. These two features can be new signs for 18p deletion syndrome.

Genomic amplification of BCR-ABL1 fusion gene and its impact on the disease progression mechanism in patients with chronic myelogenous leukemia

Identification of BCR-ABL1 fusion gene amplification status is critically important in the effective management of chronic myelogenous leukemia (CML) patients. Earlier reports suggested that overexpression of BCR-ABL1 either through amplification of BCR-ABL1 fusion gene or by the up regulation of BCR-ABL1 transcript level might be an early phenomenon in the establishment of IM resistance and disease evolution in CML. In the current study, we performed dual color dual fusion locus specific BCR/ABL1 FISH analysis along with karyotype analysis using GTG banding (G-banding using trypsin and Giemsa) technique in 489 patients with different clinical stages of CML at diagnosis or during the course of the disease to unravel the spectrum of BCR-ABL1 fusion gene amplification status. Among the study group analyzed, it was found that prevalence of occurrence of BCR-ABL1 fusion gene amplification was significantly higher in advanced stages of disease and in IM resistant CML-CP patients when compared to initial stage of disease, de novo CML-CP. Cytogenetic and metaphase FISH characterization on our study samples revealed that BCR-ABL1 fusion gene amplification was occurred through the formation of extra copies Ph chromosomes and isoderived Ph chromosomes. Current study suggests that unrestrained activity of BCR-ABL1 played a vital role in resistance to targeted therapy and disease evolution in CML. In our study population, patients in progressive stage CML and in IM resistant CP with multiple copies of BCR-ABL1 fusion gene displayed a poor response to targeted treatment with IM. Hence, the early identification of BCR-ABL1 fusion gene amplification using FISH technique will lead to improved interventions and outcome in future CML patients.

Banding cytogenetics of chimeric hybrids Coturnixcoturnix × Coturnixjaponica and comparative analysis with the domestic fowl.

The Common quail Coturnixcoturnix Linnaeus, 1758 is a wild migratory bird which is distributed in Eurasia and North Africa, everywhere with an accelerating decline in population size. This species is protected by the Bonn and Berne conventions (1979) and by annex II/1 of the Birds Directive (2009). In Algeria, its breeding took place at the hunting centre in the west of the country. Breeding errors caused uncontrolled crosses between the Common quail and Japanese quail Coturnixjaponica Temminck & Schlegel, 1849. In order to help to preserve the natural genetic heritage of the Common quail and to lift the ambiguity among the populations of quail raised in Algeria, it seemed essential to begin to describe the chromosomes of this species in the country since no cytogenetic study has been reported to date. Fibroblast cultures from embryo and adult animal were initiated. Double synchronization with excess thymidine allowed us to obtain high resolution chromosomes blocked at prometaphase stage. The karyotype and the idiogram in GTG morphological banding (G-bands obtained with trypsin and Giemsa) corresponding to larger chromosomes 1–12 and ZW pair were thus established. The diploid set of chromosomes was estimated as 2N=78. Cytogenetic analysis of expected hybrid animals revealed the presence of a genetic introgression and cellular chimerism. This technique is effective in distinguishing the two quail taxa. Furthermore, the comparative chromosomal analysis of the two quails and domestic chicken Gallusgallusdomesticus Linnaeus, 1758 has been conducted. Differences in morphology and/or GTG band motifs were observed on 1, 2, 4, 7, 8 and W chromosomes. Neocentromere occurrence was suggested for Common quail chromosome 1 and Chicken chromosomes 4 and W. Double pericentric inversion was observed on the Common quail chromosome 2 while pericentric inversion hypothesis was proposed for Chicken chromosome 8. A deletion on the short arm of the Common quail chromosome 7 was also found. These results suggest that Common quail would be a chromosomally intermediate species between Chicken and Japanese quail. The appearance of only a few intrachromosomal rearrangements that occurred during evolution suggests that the organization of the genome is highly conserved between these three galliform species.

Genetic causes of reproductive problems in the Bosnian women population

Background: The causes of infertility and recurrent spontaneous abortions are diverse and numerous – including non-genetic and genetic factors – whereby the importance of genetic factors in pathogenesis of infertility is becoming more and more common. Chromosomal abnormalities and genetic defects can cause reproduction failures, and for this reason genetic analysis can play an important role in reproductive problems research.Aims and Objective: This study aims to determine the type and frequency of chromosomalaberrations in the female population sample, as well as to determine if the difference between groups with and without chromosomal aberrations was statistically significant.Materials and Methods: One hundred women aged 15-46 were included in the study, allhaving different reproductive disorder diagnoses and requiring karyotype analysis in the Sarajevo Medical Faculty Genetic Center. Cytogenetic analysis was performed on the peripheral blood, which was cultured for four days, using GTG banding forchromosomalanalysis.Results: Out of 100 women included in the study, an abnormal karyotype was found in 16 of them (16%). The difference between the frequency of normal and abnormal karyotype in women with reproductive problems identified in this study was found to be statistically significant. The pattern of chromosomal aberrations was similar to that reported in the previous cytogenetic studies with similar inclusion criteria.Conclusion: This fact should be taken in the consideration in order to estimate true etiology of reproductive problems and it is a valuable information in the process of genetic counseling and decision making in assisted reproductive technology.Asian Journal of Medical Sciences Vol.9(5) 2018 12-16

Genotype/Phenotype correlation with partial duplications of 6p and 6q

Partial trisomy 6p and 6q are rare and clinical features include craniofacial changes, mental retardation and developmental delay. The study aims to correlate the genotype/ phenotype of a 6p and 6q duplication carrier. MVSN, female, single daughter of nonconsanguineous and healthy young couple. From an earlier relationship, the father had a 13-year-old son. Delivered by C-section, gestation of 37 weeks. The clinical analysis revealed: microcephaly, brachycephaly, syndromic facies, prominent forehead, hyperarousalism, strabismus, short nose, anteverted nares, smooth philtrum, thin lip, upper lip tented, micrognathism, dental anomalies, low-set ears, slender fingers, nails malformation, digitiform thumb, bilateral flat foot, hypotonia and neuropsychomotor developmental delay (NPMD). In the physical examination performed at 2 years of age, weight was 7,570 Kg (<3,58%), length was 74 cm (<3,95%) and cephalic perimeter was 43 cm (<2%). Classic cytogenetic techniques were performed (GTG banding and high resolution GTG), molecular cytogenetic (FISH) and cytogenetic (aCGH). The karyotype showed a mosaicism 47, XX, + mar [9]/46, XX [11] (ISCN, 2016). The parental karyotype was normal. The chromosomal changes found by aCGH indicate a genetic gain at chromosomal regions 6p11.2?q12 and 6q14.1?q14.3 with a size of 10.335 Mb and 10. 765 Mb, respectively. The patient presented mosaicism of a marker chromosome (sSMC) identified by the technique of FISH as coming from chromosome 6. In region 6p11.2?q12 the genes present are: PRIM2, GUSBP4, MTRNR2L9, KHDRBS2, LGSN, PTP4A1, PHF3, EYS and MCART3P. Heart defects and kidney problems were cited in the literature as being characteristic of trisomy 6p. However, patient did not present any of these phenotypic changes. The region 6q14.1?q14.3 contains more than 20 genes. The clinical signs found in patient such as: developmental delay, mental retardation, forehead prominent, short nose and thin lips has been described in other studies in the literature as characteristic of 6q trisomy. The presence of mosaicism excludes the possibility of sSMC as the sole responsible for the patient's phenotype. Our study correlates the phenotypic characteristics presented by the patient with partial trisomy 6q collaborating for family genetic counseling.

Fragile X syndrome: diagnosis by molecular characterization of FMR1 gene and clinical correlation

Background One of the most common forms of inherited intellectual disability (ID) is fragile X syndrome (FXS), which is caused by expansion of cytosine–guanine–guanine trinucleotide repeat at the 5′ untranslated region of the fragile X mental retardation gene (FMR1) at Xq27. The cytosine–guanine–guanine repeat expansion leads to hypermethylation and inactive transcription of the gene. The present study aimed to detect expected FMR1 gene alleles by methylation-sensitive PCR and its clinical correlation for rapid screening of FXS among male patients with ID. Patients and methods The study included 50 male patients with ID and clinical features suggestive of FXS, who were compared with 50 healthy age-matched volunteers. All patients were subjected to full history taking, thorough clinical examination using a 15-item checklist [physical (big ears, joint hyperextensibility, Simian crease, wide forehead, macroorchidism, and elongated face) and neurological features (mental retardation, family history of mental retardation, poor eye contact, hand biting, hyperactivity, perservative speech, tactile defensiveness, hand flapping, and short attention span)], and karyotyping using GTG banding. Methylation-sensitive PCR technique after bisulfite treatment of DNA was applied for the detection of expanded alleles of the FMR1 gene. Results Clinical score in patients with abnormal alleles was significantly higher compared with patients with normal alleles. GTG-banding technique showed normal 46XY male karyotype for studied patients. Frequency of normal FMR1 gene alleles was detected in the control group (100%) and 44 (88%) patients. Abnormal alleles were detected in six (12%) patients: three (6%) patients with full mutation (FM) and three (6%) patients with premutation carrier. Conclusion Our study revealed that PCR-positive results of fragile X correlate with the checklist clinical score rather than a single clinical entity.

Association of SNCA in late-onset Parkinson’s disease with 22q11.2 deletion

Parkinson’s disease (PD) is a neurodegenerative, age-related type of movement disorder, which occurs due to two pathological hallmarks such as the degeneration of the dopamine and Lewy body formation. The clinical manifestations of PD are tremor, bradykinesia, rigidity and impairment of postural reflexes. The incidence is about 17 per 100,000 person-years but in India, studies have been based on population deter- mining the exact evidence and prevalence of PD. The intend of the study was to identify the chromosomal aberrations and mutational analysis in SNCA (α-synuclein) gene in 19 late-onset of PD using GTG banding and PCR-RFLP techniques. The resulted polymorphism was observed with a protein change p.Ala53Glu (A53E) whereas the chromosomal abnormalities were observed in chromosome 22 with a deletion in 22q11.2. Therefore, these findings shows that the chromosomal aberrations and mutation in SNCA are rare in late-onset cases of PD hence, this is the foremost report in Coimbatore population and a unique study from the researches done earlier.

Down syndrome: a cytogenetic study in North Indian population

Down syndrome constitutes the most common chromosomal abnormality among live births (1 in 730 live births) and most frequent form of intellectual disability. Genetic cause for this syndrome is trisomy of chromosome 21. The cytogenetic profile of Down syndrome includes free trisomy 21, Robertsonian translocations, mosaicism, duplication of the DS critical region and other structural rearrangements involving chromosome 21. Purpose of the study was to confirm the clinical diagnosis of suspected cases of Down syndrome by karyotyping and to evaluate several risk factors associated with trisomy 21 among patients belonging to Lucknow region. Karyotype analysis was done in 46 patients with clinical suspicion of Down syndrome. GTG banding was done according to standard protocols. Findings: out of 46 cases i.e., 43 (93.5%) had abnormal karyogram. All these 43 cases had trisomy 21 (Down's syndrome). Among patients with Down syndrome, free trisomy (n=40; 93%) was most common followed by Robertsonian translocation (n=2; 4.7%) and mosaic trisomy (n=1; 2.3%) respectively. Majority of patients were males (n=27; 62.7%) and there were 16 (37.3%) females. Male to female ratio of Down syndrome patients was 1.68. 50% of patients with Down syndrome were born to mothers falling in age group of 36-40 y. Identification of various types of chromosomal abnormalities in Down syndrome is very important. It aids in management of these children and to aware the affected families about recurrence risk and options available.

Prognostic Implications of Derivative Chromosome 9 Deletions in Patients with Advanced-Stage Chronic Myelogenous Leukemia.

Elucidation of cryptic BCR/ABL1 gene rearrangement is exceptionally important in the clinical diagnosis and prognosis of chronic myelogenous leukemia (CML). Previous reports indicated an adverse prognostic effect of atypical BCR/ABL1 gene rearrangements with submicroscopic ABL1-BCR deletions on derivative chromosome 9 [der(9)] in CML patients. Dual color dual fusion locus-specific BCR/ABL1 fluorescent in situ hybridization (FISH) analysis together with G-banding using trypsin and Giemsa (GTG banding) was performed in 489 patients at different stages of CML to investigate the spectrum of BCR/ABL1 gene rearrangements. Among the study group analyzed, a significantly higher frequency of BCR/ABL1 gene rearrangements that is consistent with der(9) deletion were observed in the blast crisis (BC) phase at 41.67%, followed by the accelerated phase (AP) at 36.84%, the imatinib mesylate (IM)-resistant chronic phase (CP) at 23.08%, and the lowest incidence was found in de novo CP at 16.61%. 1R1G1F (1 red, 1 green, 1 fusion) with concurrent loss of ABL1-BCR fusion gene on der(9) chromosome was the major signal pattern identified in each group. The results from the current study show that this unusual BCR/ABL1 gene rearrangement is one of the steering forces toward disease progression in CML. Patients in AP/BC of CML with der(9) deletion showed poor response to IM therapy; however, patients with der(9) deletion in the early phases of CML responded well to IM treatment. Therefore, the establishment of an atypical FISH signal pattern in CML is of paramount important because it is associated with adverse clinical prognostic implications in advanced stages of the disease.

Challenging Reproductive Genetic Counseling in Families at High Risk for Rare Genetic Syndromes – A Case Report with Possible Denys-Drash Syndrome

Calculating the recurrence risk is an integral part of everyday practice of the genetic counselor, but certain difficulties occur when explaining it to the parents of a child with a rare dysmorphic syndrome. We present a case of a family with first newborn with multiple anomalies and exitus letalis, registered on the second day. At birth, the baby presented with limb malformations. Additionally, a spontaneous pneumothorax, neonatal cerebral ischemia, and intraamniotic infection were diagnosed. A post-mortem examination revealed additional multiple internal malformations such as patent foramen ovale, bicornuate uterus, accessory spleen, bilateral adrenal hyperplasia, unilateral Wilm’s tumor of the left kidney, diffuse glomerular and interstitial fibrosis, and protein cylinders in dilated renal tubules. Standard GTG banding karyotyping showed a normal male karyotype 46,XY,16qh+ that did not correspond to the female phenotype of the patient’s genitalia. Postmortem findings together with sex phenotype/genotype discordance suggested Denys-Drash syndrome as possible diagnosis. Amplification of all exons but exon 9 of WT1 gene was not successful on DNA extracted from formalin-fixed paraffin-embedded tissue (FFPET) sample. Sanger sequencing of exon 9 did not reveal a causative mutation. In current case the severe neonatal presentation and absence of molecular conformation complicated giving a certain prognosis for the recurrence risk. Reproductive genetic counseling in families with rare genetic conditions could be very challenging.

Frequency of down syndrome: an experience of a tertiary care diagnostic laboratory in India

Background: Down syndrome or Trisomy 21 is a genetic condition involving the presence of extra copy of chromosome 21. It is the most common chromosomal abnormality within paediatric age group. The objective of our study was to determine the frequency of Down syndrome and its various cytogenetic types in cases with clinical suspicion of Down syndrome received at the Department of Cytogenetics, Metropolis Healthcare Limited, Mumbai, India.Methods: Our study was performed on peripheral blood (2-3 ml) collected in Sodium Heparin Vacutainers obtained from 714 patients with clinical suspicion of Down syndrome. All the samples were requested for GTG staining and banding, while the cultures were set and analysed by GTG–banding at 450-550 band level. The period of our study was from January-2015 to December-2016.Results: Out of 714 samples referred, about 657 showed trisomy of chromosome 21. While, out of 657 cases, 551 (83.87%) cases were detected with free trisomy, Robertsonian translocation in 52 cases (7.91%), Mosaic pattern in 16 cases (2.44%). Our study also recorded trisomy with additional polymorphic variation in 35 cases (5.33%) and 3 cases (0.46%) with additional abnormality.Conclusions: According to the extensive literature available which states that the clinical diagnosis of Down syndrome is relatively easy, it is the pattern of chromosomal aberration that is extremely important. Identification of this pattern will assist in the estimation of the possibility of recurrence risk while counselling the parents. Overall, it will benefit the couple to arrive at an informed decision and will eventually minimize the frequency of disease in the society. Moreover, it will also assist the close blood relatives to know their risk of having baby with Down syndrome. It is to be noted that since the study was performed in a tertiary care laboratory, the percentage of cytogenetic.

Frequency of turner syndrome: findings from a tertiary healthcare diagnostic laboratory of India

Background: Turner syndrome (TS) is the most common chromosomal abnormality reported in the females. Objective of this retrospective study was to determine the frequency of turner Syndrome and its various cytogenetic types in samples suspected to be of turner syndrome, received in the Department of Cytogenetics, Metropolis Healthcare laboratory, Mumbai, Maharashtra, India. Methods: The current study was performed on 935 clinically suspected samples with Turner Syndrome within the age group of 01-16 years. Peripheral blood (2-3 ml) in Sodium heparin Vacutainers was collected from all the patients, the cultures were set & analysed by GTG–banding at 450-550 band level Results were reported as per the guidelines of the International System for Human Cytogenomic Nomenclature (ISCN) and The College of American Pathologists (CAP) and National Accreditation Board for Testing and Calibration Laboratories (NABL).Results: In our study, out of the total 935 samples referred to Metropolis Healthcare Ltd, about 348 had cytogenetically turner or Turner variant findings. Further, out of 348 cases, 69 cases were detected to have presence of single X chromosome (19.83%), mosaic pattern in 116 cases (33.33%), presence of Y chromosome in 63 cases (18.10%) polymorphic variation in 58 cases (16.67%), presence of only isochromosome Xq in 9 cases (2.59%) and 33 cases (9.48%) with other or additional abnormalities. Conclusions: The cytogenetic confirmation and pattern of chromosomal aberration is very important as early detection may help to improve the quality of life especially in patients with cytogenetically Turner variant pattern with presence of Y chromosome.

TISSUE-ENGINEERED BONE FOR TREATMENT OF COMBAT RELATED LIMB INJURIES

Based on our preliminary positive clinical results with use of cultured bone marrow-derived multipotent mesenchymal stem/stromal cells in traumatology, our aim was to develop living three-dimensional tissue-engineered bone equivalent transplantation technology for restoration of critical sized bone defects caused by combat related high energy trauma. To fabricate bone equivalent we used devitalized allogeneic bone scaffolds (blocks and chips) seeded with cultured autologous cells: bone marrow-derived multipotent mesenchymal stem/stromal cells in mix with periosteal progenitor cells and endothelial progenitor cells. Quality/identity of cell cultures was assured by donor and cell culture infection screening (immunofluorescence assay, polymerase chain reaction), flow cytometry (cell phenotype), karyotyping (GTG banding), functional assays (colony forming units analysis, multilineage differentiation assay). Bone defect treatment with bone equivalent application was fully completed in 39combat-injured with 42defects. New bone formation was assessed by the radiographic examination. Casualties were included in a treatment program an average of 10.1months after injury, provided the ineffectiveness of conventional surgery methods. All cell type cultures had a normal karyotype and appropriate phenotype, differentiation potential and functional properties, ~30% colony forming units frequency and hadn't any signs of cell senescence. The fluorescein diacetate/propidium iodide combined staining and histology analysis of graft samples before transplantation showed their regular seeding with viable cells. Pathomorphological analysis of bone equivalent specimens 3-6months post-op revealed the active remodeling processes and immature bone tissue formation. Bone defect restoration was observed 5-6months post-op. The developed biotechnology of living three-dimensional tissue-engineered bone equivalent transplantation with overall effectiveness 90.4% allows restoring the bone integrity, forming new bone tissue in a site of bone defect, and significantly reducing the rehabilitation period of a patient.

The changes of multipotent mesenchymal stromal cells isolated from human adipose tissue during long-term cultivation

Multipotent mesenchymal stromal cells (MMSCs) isolated from human adipose tissue have been subcultured for over 50 population doublings. In three cultures that experienced more than 50 cellular doublings, there appeared cells lacking contact inhibition upon reaching the monolayer. Karyotype analysis (GTG banding) showed a normal diploid karyotype, and aneuploidy and restructurings were not registered. Flow-cytometric analysis of 20 surface antigens on MMSCs in early and late passages revealed changes in the share of cells positively stained with antibodies against CD10 (a zinc depended metalloproteinase); CD34 (sialomucin); CD49 a, d, f, (α1, α4, α6, integrins); and CD71 (a transferrin receptor). Long-term cultivation influenced cell adhesion to proteins of the extracellular matrix (ECM), such as fibronectin and laminin (ligands to α, integrins), as well as the functional abilities of MMSCs to form cells of adipose and bone tissues in vitro. These findings extend our knowledge of cell behavior in culture and allow us to get closer to a deeper understanding of the processes happening to precursor cells in vitro.

A novel mosaic complex supernumerary marker chromosome in a girl with seizures: systematic characterization of the complex marker

Small supernumerary marker chromosomes (sSMC) are a heterogeneous group of chromosomes which are reported in variable phenotypes. They are chromosomal fragments or markers whose origins often cannot be determined by conventional cytogenetic methods alone and require molecular approaches. In general sSMC are equal in size or smaller than chromosome 20. The complex sSMC (CsSMC) arise from two different chromosomes. The aim of the present study is to identify and characterize the chromosomal abnormality in an 11year old girl with seizures, intellectual disability and developmental delay. Different methods like GTG banding, C-banding and NOR staining, spectral karyotyping (SKY), fluorescence in-situ hybridization (FISH) using whole chromosome paint probes (WCP) and bacterial artificial chromosome (BAC) clones were used. Also array CGH was done to check the gains and losses in the genome. The chromosomal analysis on the metaphases revealed a karyotype of mos 47,XX,+mar/46,XX. The marker was identified by SKY and aCGH, as a 33Mb duplication of the 3q region and a 38Mb duplication of the chromosome 9p region. This was confirmed by WCP FISH. Further fine mapping was done with 8 BAC clones from the 3q and 9p regions to confirm the marker chromosome. The marker showed the centromeric 9 region which was also confirmed by the BAC clones. The application of the combined cytogenetic methods like aCGH and FISH helped in the systematic characterization of the CsSMC. The accurate characterization of the CsSMC helps in increasing the knowledge of chromosomal origin, gene content, UPD and other imbalance in the genome.

COMPARISON OF CHROMOSOMAL REARRANGEMENTS IN BONE MARROW CELLS AND BLAST TRANSFORMED B-CELLS IN RELAPSE OF B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA/ SMALL LYMPHOCYTIC LYMPHOMA

The genetic mechanisms of resistance to chemotherapy in B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL) are not clear. We aimed to determine the peculiarities of abnormal karyotype formation in bone marrow (BM) cells and peripheral blood (PB) blast transformed B-cells in relapse of B-CLL/SLL. Cytogenetic GTG banding technique and molecular cytogenetic in interphase cells (i-FISH) studies of BM cells and PB blast transformed B-lymphocytes were performed in 14patients (10males and 4females) with B-CLL/SLL. The results of karyotyping BM and PB cells revealed the heterogeneity of cytogenetic abnormalities in combined single nosological group of B-CLL/SLL. In PB B-cells, chromosome abnormalities related to a poor prognosis group were registered 2.5times more often than in BM cells. Additional near tetraploid clones that occurred in 57.1% cases were the peculiar feature of BM cell karyotypes. Chromosomal rearrangements characteristic of the group of adverse cytogenetic prognosis were revealed in all cases from which in 2cases by karyotyping BM cells, in 6cases in PB B-cells and in 8cases by the i-FISH method in BM cells, i.e. their detection frequency was 3times higher in PB B-cells and 4times higher when analyzing by i-FISH in BM cells. Mismatch in abnormal karyotypes in BM and PB B-cells by the presence of quantitative and structural chromosomal rearrangements may be indicative of simultaneous and independent processes of abnormal clone formation in the lymph nodes and BM hematopoietic cells. Accumulation the information about previously unidentified chromosomal rearrangements in relapse of the disease may help to understand the ways of resistance formation to chemotherapy.