Identification of Chimeric NTRK3 Genes in Papillary Thyroid Cancer Cells by Analyzing the Imbalance of the Expression of 5' and 3' mRNA Fragments.

The standard for detecting chimeric genes of neurotrophic receptor tyrosine kinases (NTRK) is next generation sequencing (NGS). However, this analysis is expensive and takes several days. As a rapid screening method for the detection of NTRK3-dependent papillary thyroid cancer, an analysis of the expression imbalance between 5' and 3' NTRK3 mRNA fragments was used (5'/3' RT-PCR). The reference method for detection of NTRK3 rearrangements was fluorescent in situ hybridization (FISH), and the most frequent rearrangements in papillary thyroid cancer were tested using reverse transcription PCR (RT-PCR). Using 5'/3' RT-PCR, 18 samples of papillary thyroid cancer carrying chimeric transcripts of NTRK3 mRNA were detected. The sensitivity of the developed technique was 88.9% and specificity was 99.3%. Thus, a fast and cost-effective method of screening samples of papillary thyroid cancer in paraffin blocks is proposed with acceptable sensitivity and specificity.

[Epidemiology and diagnosis of mutations in the ALK gene in patients with non-small cell lung cancer in the Moscow region]. / Epidemiologiya i diagnostika mutatsii v gene ALK u patsientov s nemelkokletochnym rakom legkogo v Moskovskoi oblasti.

AIM OF STUDY: To determine a diagnostic algorithm for detecting translocation of the ALK gene and its frequency in the Moscow region. MATERIALS AND METHODS: During the priod between 2014 and 2018 (inclusive), 488 patients without activating mutations in the EGFR gene in the Moscow region were tested. To detect translocation of the ALK gene, fluorescence in situ hybridization (FISH) methods, an immunohistochemical method, and, in some cases, a polymerase chain reaction were used. RESULTS: Revealed ALK gene rearrangement in a population of patients with lung adenocarcinoma amounted to an average of 7.6% of cases. With this, the main method that we used was immunohistochemical method, applicable in more than 80% of cases. The use of other methods for verification of abnormalities in the ALK gene was found necessary in rare cases (3.3%). CONCLUSIONS: Using the algorithm presented in the article, it was possible to detect ALK gene rearrangement in a population of patients with lung adenocarcinoma in the Moscow region in an average of 7.6% of cases.

[Genomic instability in the brain: chromosomal mosaicism in schizophrenia]. / Genomnaya nestabil'nost' v kletkakh golovnogo mozga: khromosomnyi mozaitsizm pri shizofrenii.

AIM: Experimental verification of the hypothesis about the possible involvement of the mosaic genome variations (mosaic aneuploidy) in the pathogenesis of a number of mental illnesses, including schizophrenia and autism: a genetic study of the level of mosaic genome variations in cells of the brain autopsy tissues in healthy controls and schizophrenia. MATERIAL AND METHODS: Autopsy brain tissues of 15 unaffected controls and 15 patients with schizophrenia were analyzed by molecular cytogenetic methods to determine the frequency of chromosomal mutations (the mosaic aneuploidy) in neural human cells. The original collection of chromosome-enumeration DNA probes to autosomes 1, 9, 15, 16, 18 and the sex chromosomes X and Y was used for the interphase cytogenetic analysis of chromosomes in the cells of the brain. RESULTS AND CONCLUSION: The frequency of low-level aneuploidy per individual chromosome was 0.54% (median - 0.53%; 95% confidence interval (CI) CI - 0.41-1.13%) in controls and 1.66% (median - 1.55%; 95% CI -1.32-2.12%) in schizophrenia (p=0.000013). Thus, the three-fold increase in aneuploidy frequency in the brain in schizophrenia was detected. It is suggested that mosaic aneuploidy, as a significant biological marker of genomic instability, may lead to genеtic imbalance and abnormal functional activity of neural cells and neural networks in schizophrenia.

[Clinical and genetic characteristics of the X chromosome distal long arm microduplications encompassing the MECP2 gene].

OBJECTIVE: Microduplications of the long arm of the X chromosome including the MECP2 gene are relatively common causes of neurodevelopmental disorders in males. Authors analyzed clinical presentations of this disease in children. MATERIAL AND METHODS: Authors performed a clinical and genetic analysis of four cases using contemporary cytogenetic, molecular cytogenetic studies (FISH, array CGH) and X chromosome inactivation analysis. RESULTS AND CONCLUSION: We described somatic, neurologic and mental symptoms of the patients. The genetic imbalance impact on the patients' phenotype, necessity of comprehensive family studies for correct genetic diagnosis and effective genetic counseling in cases of microduplications of the long arm of the X chromosome including the MECP2 gene are discussed.

[Use of the new 2013 ASCO/CAP guidelines to study HERS status by fluorescence in situ hybridization in patients with breast cancer].

UNLABELLED: Despite the long-term history of HER2 testing in breast cancer (BC), the results of its study are frequently interpreted ambiguously. The introduction of the new 2013 ASCO/CAP guidelines is aimed to further optimize HERS testing in view of new scientific evidence for the biological characteristics of BC. OBJECTIVE: to study the results of assessing HERS2 status in patients with the questionable level of protein expression according to the 2007 versus 2013 ASCO/CAP criteria. SUBJECTS AND METHODS: Biopsy specimens from 572 patients diagnosed with BC after immunohistochemical examination, including HER2 status assessment, were investigated by fluorescence in situ hybridization. RESULTS: The use of the new 2013 ASCO/CAP criteria versus the previous criteria to determine intratumor heterogeneity, HER2 gene copy number, and the so-called chromosome 17 polysomy was ascertained to cause a statistically significant increase in HER2-positive cases from 25 to 42.5% (OR=2.2178; 95% CI, 1.8254-2.6950; р=0.0005). CONCLUSION: The use of the new guidelines additionally permits effective therapy in patients with BC, which may be another step to improve survival in this large patient group.

[The use of molecular cytogenetic and cytogenetic techniques for the diagnosis of Prader-Willi and Angelman syndrome].

We examined 30 patients with a presumptive diagnosis of Prader-Willi and Angelman syndromes. In four patients, 15q11.2-q13 deletions were identified by cytogenetic techniques. The FISH method was used to study eight patients, in five of them microdeletions were also confirmed. High-resolution comparative genomic hybridization (CGH) and comparative genomic hybridization using DNA microarrays (array CGH) allowed to find 15q11.2-q13 deletions in five patients. These cases demonstrate the need for high-resolution post-genomic technologies (array CGH - molecular karyotyping) in the combination with classical cytogenetic and molecular cytogenetic techniques.

[The detection of activating somatic mutations in gene KRAS using pyrosequencing technique].

The technique to detect all possible variants of mutations in 12, 13 and 15 codons of gene KRAS was developed on the basis of the pyrosequencing technology. The analytical characteristics of the developed technique were identified. The limit of detection for mutations G34T, G35A and G38A detected on the cloned control samples consisted 3%. The limit of blank for various mutations consisted from 0.3% to 4.1%. The system was tested on clinical samples. The 7 different types of mutations were identified and detected in quantitative format. No discrepancy of pyrosequencing data with results of sequencing according Sanger was established.

[Subchromosomal microdeletion identified by molecular karyotyping using DNA microarrays (array CGH) in Rett syndrome girls negative for MECP2 gene mutations].

Molecular karyotyping using DNA microarrays (array CGH) was applied for identification of subchromosomal microdeletions in a cohort of 12 girls with clinical features of RETT syndrome, but negative for MECP2 gene mutations. Recurrent microdeletions of MECP2 gene in chromosome X (locus Xq28) were identified in 5 girls of 12 studied. Probably RTT girls with subchromosomic microdeletions in Xq28 could represent a special subtype of the disease, which appears as clinically milder than the classic form of disease. In one case, an atypical form of RTT was associated with genomic abnormalities affecting CDKL5 gene and region critical for microdeletion Prader-Willi and Angelman syndromes (15q11.2). In addition, data are presented for the first time that genetic variation in regions 3p13, 3q27.1, and 1q21.1-1q21.2 could associate with RTT-like clinical manifestations. Without application of molecular karyotyping technology and bioinformatic method of assessing the pathogenic significance of genomic rearrangements these RTT-like girls negative for MECP2 gene mutations were considered as cases of idiopathic mental retardation associated with autism. It should be noted that absence of intragenic mutations in MECP2 gene is not sufficient criteria to reject the clinical diagnosis of RTT. To avoid errors in the genetic diagnosis of this genetically heterogeneous brain disease molecular cytogenetic studies using high resolution oligonucleotide array CGH (molecular karyotyping) are needed.

Cytogenetic, molecular-cytogenetic, and clinical-genealogical studies of the mothers of children with autism: a search for familial genetic markers for autistic disorders.

State-of-the-art cytogenetic and molecular-cytogenetic methods for studying human chromosomes were used to analyze chromosomal anomalies and variants in mothers of children with autistic disorders and the results were compared with clinical-genealogical data. These investigations showed that these mothers, as compared with a control group, showed increases in the frequencies of chromosomal anomalies (mainly mosaic forms involving chromosome X) and chromosomal heteromorphisms. Analysis of correlations of genotypes and phenotypes revealed increases in the frequencies of cognitive impairments and spontaneous abortions in the mothers of children with autism with chromosomal anomalies, as well as increases in the frequencies of mental retardation, death in childhood, and impairments to reproductive function in the pedigrees of these women. There was a high incidence of developmental anomalies in the pedigrees of mothers with chromosomal variants. These results lead to the conclusion that cytogenetic and molecular-cytogenetic studies of mothers and children with autism should be regarded as obligatory in terms of detecting possible genetic causes of autism and for genetic counseling of families with autistic children.

[Chromosomal mosaicism in spontaneous abortions: analysis of 650 cases].

It is known that up to 50% spontaneous abortions (SA) in the first trimester of pregnancy are associated with chromosomal abnormalities. We studied mosaic forms of chromosomal abnormalities in 650 SA specimens using interphase mFISH and DNAprobes for chromosomes 1,9, 13/21, 14/22, 15, 16, 18, X, and Y. Numerical chromosomal abnormalities were discovered in 58.2% (378 cases). They contained combined chromosomal abnormalities (aneuploidy of several chromosomes or aneuploidy in combination with polyploidy in the same specimen) in 7.7% (29 cases) or 4.5% of the entire SA sample; autosomal trisomy, in 45% (18.2% in chromosome 16, 8.9% in chromosomes 14/22, 7.9% in chromosomes 13/21, 3.1% in chromosome 18, and 1.4% in chromosome 9). Chromosome X aneuploidy was found in 27% cases, among which 9.6% represented chromosome X monosomy. Polyploidy was observed in 22.9% cases. In 5.1% cases, we observed mosaic form of autosomal monosomy Among the SA cases with chromosomal abnormalities mosaicism was observed in 50.3% (approximately 25% of the entire SA sample). The results of the present study indicate that significant amount of chromosomal abnormalities in SA cells are associated with disturbances in mitotic chromosome separation, which represents the most common cause of intrauterine fetal death. It was also shown that original collection of DNA probes and the technique of interphase MFISH could be useful for detection of chromosomal mosaicism in prenatal cell specimens.

[Cytogenetic, molecular cytogenetic, clinical and genealogical study of mothers of children with autism: a search for family genetic markers of autistic disorders].

Using modern cytogenetic and molecular cytogenetic techniques towards the study of human chromosomes, an analysis of chromosomal abnormalities/chromosomal variations as well as clinical and genealogical data in mothers of children with autism has been performed. It has been shown that mothers of autistic children exhibit an increased incidence of chromosomal abnormalities (mainly mosaic forms involving chromosome X) and an increased occurrence of chromosomal variations compared to controls. The analysis of genotype-phenotype correlations revealed the increase in the frequency of cognitive disturbances and spontaneous abortions in mothers of children with autism as well as the higher frequency of mental retardation, early death and reproductive problems in the pedigrees. The high frequency of congenital malformations in the pedigrees of mothers with chromosomal variations was observed as well. Taking into account the data obtained, we have concluded that cytogenetic and molecular cytogenetic studies of mothers of children with autism are obligatory for detection of possible genetic causes of autism and genetic counseling of families with children affected with autistic disorders.

[Transplantations of allogenic and autologous hemopoietic stem cells in acute leukemia (results of 20-year experience)].

AIM: To analyse results of transplantation of allogenic and autologous hemopoietic stem cells (allo-THSC and auto-THSC) with myeloablation preconditioning in patients with acute leukemia (AL) performed in 1987-2006. MATERIAL AND METHODS: A total of 71 allogenic and 45 autologous THSC were performed in 116 patients with different AL variants. Conditioning in all allo-THSC included busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg). This regimen was used in 29 recipients of auto-HSC. Cyclophosphamide in a dose 120 mg/kg and total radiation of the body in a dose 12 Gy were given to 16 recipients. Overall, relapse-free and event-free survival of patients after THSC were analysed as well as early (first 100 days) and overall lethality. Auto-THSC in 15 patients was for the first time followed by immunomodulating therapy aimed at prevention of AL relapses: in acute myeloid leukemia ATRA in combination with alpha-interferon, in acute lymphoblastic leukemia (ALL)--ronkoleukin, interleukin-2 preparation. RESULTS: Overall survival of AL patients after allo-THSC for the observation period increased from 31 to 58%, early lethality fell from 44 to 4%. Results of allo-THSC conducted in the first complete remission were much better than in patients with other AL stages at the time of THSC. After auto-THSC 5-year survival rose from 22 to 60% while early lethality reduced from 33 to 4%. Administration of immunomodulating therapy after auto-THSC increases 5-year survival from 35 to 80%. CONCLUSION: Outcomes of THSC in AL has improved for the last 20 years. Outcomes of allo-THSC performed in the first complete remission are much higher. Immunomodulating therapy after auto-THSC promoted better results.

[Allogenic transplantation of hemopoietic stem cells in low-intensity regimes in patients with hematological malignancies].

AIM: To test feasibility of transplantation of hemopoietic stem cells (THSC) with conditioning in low-intensity regimen associated with minimal toxic complications and engraftment in patients with hematological malignancy (HM) from a high risk group. MATERIAL AND METHODS: THSC was performed in 33 patients aged 18 to 65 years. Most of the patients suffered from acute leukemia and advanced forms of myelodysplastic syndrome. All the patients had severe complications excluding standard transplantation. Pretransplantation preparation was based on fludarabine and moderate doses of busulfane. Engraftment was achieved in 94% patients. Of complications, there were primarily infections, relapses, graft versus host reactions (45, 24, 57.5%, respectively). Overall survival was 53%, relapse-free--67%, follow-up median 23.6 months. CONCLUSION: THSC after conditioning in the regime of low intensity is an effective method of HM treatment in patients with contraindications to standard transplantation. The main problem is a high risk to develop graft versus host reaction, especially a chronic form.

Variability in the heterochromatin regions of the chromosomes and chromosomal anomalies in children with autism: identification of genetic markers of autistic spectrum disorders.

Cytogenetic and molecular cytogenetic analysis of children with autism (90 subjects) and their mothers (18 subjects) is presented. Anomalies and fragility were found in chromosome X in four cases of autism: mos 47,XXX[98]/46, XX[2]; 46,XY,r(22)(p11q13); 46,XY,inv(2)(p11.2q13),16qh-; and 46,Y,fra(X)(q27.3),16qh-. C staining and quantitative fluorescent in situ hybridization (FISH) were used to demonstrate a significant increase in the frequency of variations in the heterochromatin regions of chromosomes in children with autism as compared with a control group (48% and 16% respectively). Pericentric chromosome inversion 9phqh was not characteristic of patients with autism, while variation in heterochromatin regions 1phqh, 9qh+, and 16qh-were found significantly more frequently in children with autism. These data provide the basis for discussing the possible role of the gene position effect in the pathogenesis of autism and the possible search for biological markers of autistic disorders.

Unexplained autism is frequently associated with low-level mosaic aneuploidy.

BACKGROUND: Autism is a common childhood neurodevelopmental disorder with a possible genetic background. About 5-10% of autism cases are associated with chromosomal abnormalities or monogenic disorders. However, the role of subtle genomic imbalances in autism has not been delineated. This study aimed to investigate a hypothesis suggesting autism to be associated with subtle genomic imbalances presenting as low-level chromosomal mosaicism. METHODS: We surveyed stochastic (background) aneuploidy in children with/without autism by interphase three-colour fluorescence in situ hybridisation. The rate of chromosome loss and gain involving six arbitrarily selected autosomes and the sex chromosomes was assessed in the peripheral blood cells of 60 unaffected children and 120 children with autism. RESULTS: Of 120 analysed boys with autism, 4 (3.3%) with rare structural chromosomal abnormalities (46,XY,t(1;6)(q42.1;q27); 46,XY,inv(2)(p11q13); 46,XY,der(6),ins(6;1)(q21;p13.3p22,1)pat; and 46,XY,r(22)(p11q13)) were excluded from further molecular cytogenetic analysis. Studying <420 000 cells in 60 controls and 116 children with idiopathic autism, we determined the mean frequency of stochastic aneuploidy in control and autism: (1) autosome loss 0.58% (95% CI 0.42 to 0.75%) and 0.60% (95% CI 0.37 to 0.83%), respectively, p = 0.83; (2) autosome gain 0.15% (95% CI 0.09 to 0.21%) and 0.22% (95% CI 0.14 to 0.30%), respectively, p = 0.39; and (3) chromosome X gain 1.11% (95% CI 0.90 to 1.31%) and 1.01% (95% CI 0.85 to 1.17%), respectively, p = 0.30. A frequency of mosaic aneuploidy greater the background level was found in 19 (16%) of 116 children with idiopathic autism, whereas outlier values were not found in controls (p = 0.0019). CONCLUSIONS: Our findings identify low-level aneuploidy as a new genetic risk factor for autism. Therefore, molecular cytogenetic analysis of somatic mosaicism is warranted in children with unexplained autism.

[Monitoring of minimal residual disease in patients with acute promyelocytic leukemia].

AIM: To study efficacy of different programs of maintenance therapy, to create the program of differential therapy of minimal residual disease (MRD) and molecular recurrences at all stages of acute promyelocytic leukemia (APL) basing on the results of monitoring. MATERIAL AND METHODS: A total of 76 APL patients entered the trial. They received therapy by the protocols APL-97/98, APL-01, AIDA, 5D. Expression of chymeric oncogen PML/RARa in the disease onset was estimated by bone marrow and/or peripheral blood examination with RT-PCR. The study of chymeric oncogen PML/RARa was made once in two months. RESULTS: The program of differential therapy of APL is proposed on the basis of molecular-biological monitoring of expression of chymeric oncogen PML/RARa. The results of molecular monitoring of MRD correlated with development of molecular and hematological recurrences. Therapeutic policy is determined after diagnosis of molecular recurrence. Further therapy of APL is determined which allows a rise in overall and recurrence-free survival of the patients. CONCLUSION: The efficacy of maintenance therapy only with cytostatic drugs or their combination with ATRA is similar. The response to biological therapy with ATRA plus interferon-alpha is not sufficient. Molecular recurrences--probable or documented--are detected in maintenance therapy 2 months earlier, on the average, than hematological ones. Changes in the treatment policy in registration of molecular recurrence significantly diminish probability of hematological recurrence (from 36 to 0%, p = 0.001.

Pericentric inversion inv(7)(p11q21.1): report on two cases and genotype-phenotype correlations.

We report on two unrelated cases of pericentric inversion 46,XY,inv(7)(p11q21.1) associated with distinct pattern of malformation including mental retardation, development delay, ectrodactyly, facial dismorphism, high arched palate. Additionally, one case was found to be characterized by mesodermal dysplasia. Cytogenetic analysis of the families indicated that one case was a paternally inherited inversion whereas another case was a maternally inherited one. Molecular cytogenetic studies have shown paternal inversion to have a breakpoint within centromeric heterochromatin being the cause of alphoid DNA loss. Maternal inversion was also associated with a breakpoint within centromeric heterochromatin as well as inverted euchromatic chromosome region flanked by two disrupted alphoid DNA blocks. Basing on molecular cytogenetic data we hypothesize the differences of clinical manifestations to be produced by a position effect due to localization of breakpoints within variable centromeric heterochromatin and, alternatively, due to differences in the location breakpoints, disrupteding different genes within region 7q21-q22. Our results reconfirm previous linkage analyses suggested 7q21-q22 as a locus of ectrodactily and propose inv (7)(p11q21.1) as a cause of recognizable pattern of malformations or a new chromosomal syndrome.

[Variations of heterochromatic chromosomal regions and chromosome abnormalities in children with autism: identification of genetic markers in autistic spectrum disorders].

In the present study, the cytogenetic and molecular cytogenetic analysis of 90 children with autism and their mothers (18 subjects) was carried out. Chromosome fragility and abnormalities were found in four cases: mos 47,XXX[98]/ 46,XX[2]; 46,XY,r(22)(p11q13); 46,XY,inv(2)(p11.2q13),16qh-; 46Y,fra(X)(q27.3)16qh-. Using C-banding and quantitative fluorescent in situ hybridization (FISH), the significantly increased incidence of heterochromatic region variation was shown in autism as compared to the controls (48 and 16%, respectively). Pericentric 9phqh inversion was not characteristic of the patients with autism whereas heterochromatic variations 1phqh, 9qh+ and 16qh- were more frequent in autism (p<0,05). Basing on the data obtained, a possible role of position effect in autism pathogenesis as well as a potential of heterochromatic region variation analysis for the search of biological markers of autistic spectrum disorders are discussed.

Chimerism and multiple numerical chromosome imbalances in a spontaneously aborted fetus.

We report on a case of chimerism and multiple abnormalities of chromosomes 21, Xand Yin spontaneous abortion specimen. To the best our knowledge the present case is the first documented chimera in a spontaneously aborted fetus. The application of interphase fluorescence in situ hybridization (FISH) using chromosome enumeration and site-specific DNA probes showed trisomy X in 92 nuclei (23 %), tetrasomy X in 100 nuclei (25 %), pentasomy of chromosome X in 40 nuclei (10 %), XXY in 36 nuclei (9 %), XXXXXXYY in 12 nuclei (3 %), XXXXXYYYYY in 8 nuclei (2 %), trisomy 21 and female chromosome complement in 40 nuclei (10 %), normal female chromosome complement in 72 nuclei (18 %) out of 400 nuclei scored. Our experience indicates that the frequency of chimerism coupled with multiple chromosome abnormalities should be no less than 1 : 400 among spontaneous abortions. The difficulties of chimerism identification in fetal tissues are discussed.