We report on results of quality for preparation of cytogenetic slides and chromosomal analysis in the laboratories of RF in the system of the interlaboratory comparative examinations «FSEQA» in 2017. Common causes of poor results of the are discussed.

Numerical and cytogenetically identifiable structural abnormalities of sex chromosomes, their unbalanced microstructural rearrangements - copy number variations (CNVs) and gene variants are a common cause of disorders of sex differentiation and reproductive organs development, hypogonadism, growth abnormalities, mental retardation, defects of gametogenesis and fertility. Due to rapid development of molecular technologies of genome analysis in recent years, data on the «fine structure» of the genome and its variations, including sex chromosomes, their recombination and mutations, and numerous CNVs were obtained. The variety of gonosomal mutations, the frequent combination of changes in different levels of the genome and the presence of sex chromosome mosaicism, also as the lack of a universal approach to their diagnosis requires a comprehensive, and in many cases, an individual algorithm of medical and genetic examination of patients with abnormalities of sex development, physical and mental development, reproductive function. The article provides a brief overview of human sex chromosomes and CNVs, as well as modern possibilities of their diagnosis.

Сhromosomal microarray (CMA) testing is a first-tier test for patients with developmental delay, autism spectrum disorder, or congenital anomalies. CMA has high diagnostic yield for those patients and can detect chromosomal aberrations of less than 5 Mb. However, copy number variations (CNVs) detected by CMA in the form of deletion and duplication (triplication) cannot always be visualized in the context of metaphase chromosomes and hence, the mechanism causing the chromosomal imbalance cannot be identified. In some cases further techniques are needed for detailed characterization of chromosomal abnormalities to understand the structure of the abnormalities. The only way to do this is by karyotyping of methaphase chromosomes, combined, if necessary with FISH. Identification of the underlying mechanism is required for genetic counseling purposes, for example for presenting an estimate of the recurrence risk to the patient’s parents. Follow-up studies after CMA in patients with developmental delay and/or congenital anomalies, suspected chromosome abnormalities leading to the formation of pathogenic CNVs and the mechanisms causing the chromosome imbalance are discussed.

Molecular genetic causes of hereditary epilepsy are diverse and include both monogenic and chromosomal pathologies. Monogenic causes of hereditary epilepsies are better studied than seizures syndromes with chromosome rearrangements. In the study of the group of patients with seizures caused by chromosome rearrangements, it was shown that the most frequent changes are those affecting the long arm of the second chromosome and the short arms of chromosome 1 and 4. The severity of the disease depends not so much on the size of the rearrangements as on the list of the genes, included in the affected area. In addition, when comparing groups of patients with chromosomal ore monogenic causes of hereditary epilepsy, it has been established that in the majority of cases, the chromosomal pathology debuts with a delay in psycho-motor development preceding the onset of seizures, while in monogenic diseases, seizures tend to occur earlier followed by the delay of development. These clinical-genetic correlations play a significant role in the order of the molecular diagnostic procedure aimed at optimizing the genetic diagnosis of hereditary epilepsies.

Background. Insulinomas are rare (1-4 cases per million people) and typically sporadic neuroendocrine tumors of the pancreas, manifesting mainly by hypoglycemia caused by endogenous hyperinsulinism. So far, only a few studies of the genomes of these tumors have been carried out, and a generally accepted idea of their etiopathogenesis at the molecular genetic level has not been developed. Hypothesis. Recently, studies in the field of insulinomas drug therapy have demonstrated the effectiveness of everolimus, an inhibitor of a protein kinase of the serine-threonine specificity mTOR. Everolimus is known as a targeted drug for the treatment of tuberous sclerosis, a key role in the etiopathogenesis of which is played by alterations of the TSC1 and TSC2 genes. We hypothesized that the high therapeutic efficacy of everolimus against insulinomas, by analogy, may be due to the pivotal involvement of mutations in the TSC1 and TSC2 genes in the development of these tumors. Material and methods. To test the hypothesis, we performed NGS of exons and adjacent introns of the TSC1 and TSC2 genes in order to identify point mutations and losses of heterozygosity in 9 pancreatic insulinomas. Result. Totally in 9 samples we have identified eight point mutations and seven cases of loss of heterozygosity. In 4 samples, the tumor genotype corresponds to a two-hit tumorigenesis model (two different mutational events in the TSC2 gene). In two samples, losses of heterozygosity encompassing simultaneously the TSC1 and TSC2 genes were identified. In only one case, no point mutations nor losses of heterozygosity in the TSC1 or TSC2 genes were found. Conclusion. This is the first report of a high occurrence of mutations in the genes of the tuberous sclerosis complex in insulinomas of the pancreas, indicating possible involvement of the TSC1/TSC2 protein complex disruption in the development of most tumors of this type. Further research should lead to a deeper understanding of the causes of pancreatic neuroendocrine tumors and to the improvement of their treatment, including that with mTOR inhibitors.

Introduction: Copy number variation is regarded as one of the leading genetic causes of intellectual disorders. About 230 microdeletions and only about 80 microduplication syndromes are described in patients with intellectual disability and developmental disorders. In connection with the prevalence of microdeletions with proven pathogenetic significance, the question arises about underestimation of the role of microduplications in the realization of pathological conditions. Aim: search for pathogenetically significant chromosomal microduplications in patients with idiopathic intellectual disability and characterization of their clinical manifestations. Materials and Methods: Using 8х60K arrays the molecular karyotyping for 200 children of 2-18 years old with developmental delay, intellectual disability (IQ 50-70), and dysmorphic features / congenital anomalies was performed. Verification and analysis of the origin of microduplications were carried out using PCR-RT. Results: Seventy patients (35%) showed no clinical significant CNV, and 63 (31.5%) reported benign variants. A wide spectrum of pathogenic and potentially pathogenic CNVs was identified for 67 patients (33.5%), not detected with standard karyotyping. In 39 children of CNVs with probable pathogenetic significance, 14 deletions and 25 duplications were identified, which in the general group of patients consist of 7% and 12.5%, respectively. Eighteen patients (9%) had combinations of different types of CNVs (with pathogenic and potentially pathogenic significance). Ten patients (5%) were diagnosed with a microdeletion / microduplication syndrome. Conclusions: We determined the frequency, spectrum, origin of clinically significant chromosomal microduplications in patients with idiopathic intellectual disability and characterized its clinical manifestations. Timely molecular cytogenetic diagnosis is important for elucidating the causes of the disease and providing quality medical genetic counseling for the family.

Chromosome diseases are genetically determined diseases, manifested by a variety of symptoms. Despite the high frequency of chromosome abnormalities and their clinical significance, there are no approaches to effective treatment of patients and to the correction of large chromosome defects. Several methods for the eliminating of the mutant chromosome from the cell are proposed: through spontaneous loss of the ring chromosome during the culturing of induced pluripotent stem cells (iPSC) and by removing its centromere using the CRISPR/Cas9 system. It was shown that the ring chromosome can be created using CRISPR/Cas9 technology. We tested the first approach for ring chromosome elimination and showed that during the culturing of iPSCs the ring chromosome 13 formed stable fragments, while the ring chromosome 22 was transmitted unchanged. Obviously, the loss of the ring chromosome in iPSCs is not strictly a universal process, and the technologies that can form the basis of chromosome therapy require considerable improvement.

Background. Neoadjuvant chemotherapy (NAC) is a common practice for locally advanced breast cancer to downstage the disease to become operable. The top-of-the-line predictor of the effectiveness of neoadjuvant treatment is the pathologic complete response, defined as the absence of viable tumor cells in the mammary gland and regional lymph nodes. According to different studies, this result is achieved in no more than 13-33% of patients. Increasing the effectiveness of NACHT can be achieved through the identification of predictive markers that allow assessing the sensitivity to therapy. Objective. To assess the methylation status of SLC9A3, DPYS, IRF4, ADCY8, KCNQ2, TERT, SYNDIG1 and SKOR2 genes in tumor and serum samples and to analyze its association with response to breast cancer neoadjuvant chemotherapy. Material and methods. Core biopsy and plasma samples were obtained before and after neoadjuvant chemotherapy from 36 primary breast cancer patients. DNA methylation status was assessed using methylation sensitive PCR. Result. DNA methylation analysis of tumor biopsy material obtained before treatment showed the following gene methylation frequencies: SLC9A3 - 27.8% (10/36), DPYS - 8.3% (3/36), IRF4 - 22.2% (8/36), ADCY8 - 41.7% (15/36), KCNQ2 - 27.8% (10/36), TERT - 8.3% (3/36), SYNDIG1 - 16.7% (6/36) and SKOR2 - 5.5% (2/36). For further investigation in the blood plasma, 3 genes with the highest methylation frequencies were selected: SLC9A3 , KCNQ2 and ADCY8 . For the ADCY8 gene in the blood plasma, a statistically significant difference in methylation frequencies (p = 0.0076, exact two-sided Fisher test) was found between groups with different degrees of therapeutic response (methylation was more often observed in the group with poor response to treatment). Conclusion. Our results indicate that the methylation status of ADCY8 gene in plasma before treatment is associated with NAC pathological complete response in breast cancer.

Congenital myasthenic presynaptic syndrome type 20 - neuromuscular disease with autosomal recessive inheritance caused by homozygous or compound heterozygous mutations in the SLC5A7 gene, characterized by severe hypotonia associated with episodic apnea soon after birth of the child. This article describes the case of SLC5A7 gene mutation detection in girl (age 5 month) with respiratory failure, hypoxic-ischemic encephalopathy, severe muscle hypotonia. The previously described homozygous variant of the nucleotide sequence in the 6 exon of the SLC5A7 gene (p.Ile291Thr) was detected by whole-exome sequencing. This mutation was validated by the Sanger sequencing method in patients. The mother of the child has a mutation in the heterozygous state, the father has no the same mutation. One cannot exclude the presence of gonadal mosaicism and/or «false paternity» in the father in view of the presence of a homozygous mutation in the child and the absence of a second mutation in the parents. The presented clinical observation of a patient with a rare genetic form of myasthenia with respiratory failure shows the importance of DNA diagnostics using the method of whole-exome sequencing in order to search for a molecular defect and to determine the further option of patients management with this severe pathology.

We report on a molecular cytogenetic diagnosis of mosaicism with an unbalanced autosomal translocation and a normal cell line. Case 1. The patient was referred for evaluation because of developmental delay. The karyotype of this patient was normal. Chromosomal micro-array analysis detected a terminal deletion of the short arm of chromosome 8 and a terminal duplication of the long arm of chromosome 10. Additional FISH analysis showed an unbalanced translocation in 77% and a normal karyotype in 23% of cells. Case 2. The patient was referred for Сri-du-chat syndrome. Chromosome analysis revealed a derivative chromosome 5. FISH analysis showed a mosaic karyotype with an unbalanced translocation in 88% and a normal karyotype in 12% of cells.