Analysis of the genetic material of single cells is a very urgent task for such fields of science as oncology, forensic medicine, preimplantation genetic diagnosis. Whole genome amplification is an indispensable stage in the study of single cells. Among methods of whole genome amplification, methods based on thermocycling and isothermal methods are distinguished. Each of the methods of whole genome amplification should ensure the maximum possible representation and proportionality of all parts of the investigated genome, and also have a minimal bias, that is, it is most accurate to represent the primary DNA sequence in products of whole genome amplification. These characteristics can be influenced by the types of primers, polymerases used in the methods of whole genome amplification, as well as the initial quality and quantity of the DNA being studied. Therefore, depending on the protocol used, the quality of the amplification may vary significantly, which should undoubtedly be taken into account when choosing the method of whole genome amplification in accordance with the intended purpose of the study.

Priority in the prevention of congenital malformations and chromosomal abnormalities in children and prevention of infant mortality, disability, morbidity from this type of pathology is prenatal diagnosis (PD). The leading measure in it today is early prenatal screening (ЕPS), performed centrally at the expert level of diagnosis and allowing for 11-14 weeks with 85% sensitivity to detect frequent chromosomal trisomies (CНA) and gross anatomical defects (СМ) in the fetus. Since 2014, the Ministry of Health of the Russian Federation has been conducting an annual external quality control of the activities of the ЕPS and assesses its effectiveness according to the original data from the unified software system of the ЕPS system for all entities. In 2017, with data for 2016, 77 subjects and 3 zonal representatives took part in the audit. With an average coverage of 80% in 2016, 1,185,274 pregnant women passed through the expert diagnostic level in the RF subjects of the Russian Federation. In terms of 11-14 weeks of pregnancy, 2857 СМ and 3195 СНA were prenatally detected.

Charcot-Marie-Tooth disease (CMT) is one of the most frequent neurodegenerative disorder with a prevalence of 1 cases per 2500. CMT1 is the most common subtype of CMT, accounting for 70-80% of all cases. In the majority of cases CMT1A caused by 1.5 Mb duplication in the 17p11.2, including the peripheral myelin protein 22 ( PMP22 ) gene. Preimplantation genetic diagnosis (PGD) for this pathogenic genetic variant is a powerful tool for disease prevention. Previously, a test system for PGD CMT1A was performed by 1-5 polymorphic loci. However, the analysis of 1-5 markers with incomplete informativity cannot be considered as highly reliable under PGD conditions. This paper reports on the PGD for CMT1A in three couples using a test system with 27 polymorphic markers. The presented test system provides accurate and reliable results in the PGD for the 17p11.2 duplication and point mutations in PMP22 gene.

The results of a study of 38 female with a clinical diagnosis of «Duchenne/Becker muscular dystrophy». All probands searched for deletions / duplications DMD gene, number of sex chromosomes and X inactivation, mutations in autosomal recessive LGMD-genes: CAPN3, FKRP, SGCA, SGCB, SGCG, SGCD. The cause of muscular dystrophy was established in 45% of the examined probands. It is shown that it is impossible to confirm the diagnosis of muscular dystrophy in women on the basis of detection of unbalanced X-chromosome inactivation. The medical technology «Single tube detection system for frequency sarcoglicans mutations» was introduced into the practical activity of the Research Centre for Medical Genetics.

Congenital aniridia is a Mendelian autosomal dominant panocular disorder with complete penetrance and variable expressivity. The incidence of aniridia is 1 in 40,000-100,000 births. Aniridia is characterized by congenital absence of the iris with foveal hypoplasia and other eye abnormalities. Aniridia occurs as non-syndromic which, however, often affects all eye structures (75% of cases) and syndromic (20%, including WAGR syndrome). The most cases of aniridia, both isolated and syndromic, is caused by heterozygous mutations in the PAX6 gene or chromosome 11p13 rearrangements. Large deletions of the same region affecting PAX6 and WT1 genes loci lead to the WAGR syndrome. 110 patients with a preliminary diagnosis of congenital aniridia from 84 unrelated families and 7 patients from 7 unrelated families diagnosed with WAGR syndrome were included into the study. The applied complex confirmatory and differential DNA diagnosis of aniridia and WAGR syndrome consists of two main sequential steps: MLPA analysis (with confirmation of the detected deletions using the loss of heterozygosity analysis and/or FISH) and Sanger sequencing. The efficiency of MLPA analysis as an initial diagnostic method is 33% (30/91). The effectiveness of sequencing as the only method for diagnosis of aniridia is 63.7% (58/91). As a result of the application of complex medical technology at the Research Centre for Medical Genetics, the diagnosis was confirmed in 107 patients (81 probands) with aniridia and 7 patients with WAGR syndrome. Frequent PAX6 mutations and 11p13 deletions were identified. The effectiveness of two-staged technology for congenital aniridia diagnosis is 96.7% (88/91).

Warfarin is prescribed to a lot of patients with different pathologies, however there are individual differences of required dosage up to twenty times. Overdosage can cause health complications like different localized bleeding, up to if threatening condition. There are genetics factors, that influences on warfarin metabolism in the human body. The point of this research is the development of set of oligonucleotide and genotyping of genetic polymorphism, that influence on warfarin effect. The developed multiplex panel (мультиплексная панель) includes six polymorphic variants in biotransformation genes, and their combination determine correct warfarin dose: CYP2C9 (rs1799853, rs1057910), VKORC1 (rs9923231, rs8050894), CYP4F2 (rs2108622) and GGCX (rs11676382).Genetic replacement types and DNA characteristics in SNP region were analysed to develop the sample set. The multiplexing opportunity was checked according to the results of the reaction with Primer Focus Kit. Multiplex genotyping of DNA control samples with known genotypes was performed. Genotypes wich were identified using SNaPshot, corresponds to those, indentified using standard method. In summary, sample panel for multiplex genotyping of six SNP was developed and verified, SNP are connected with individual warfarin sensibility. Using these results in clinical practice can increase efficiency of anticoagulant therapy and decrease side effects frequency.

Symptomatic and pathogenetic treatment of cystic fibrosis allowed to increase patients’ lifespan up to 30 years, but disease is still incurable. New gene therapy technologies based on the use of specific nucleases open up new opportunities in development of etiology-based therapy for hereditary diseases. Among these methods CRISPR/Cas9 is the most widely used approach for genome editing. The aim of the study is to compare correction efficacy of CFTR gene by Cas9 with different guide RNAs (sgRNAs), designed to F508del mutation, and to increase their activity. We used modified spCas9 (eSpCas9) and two sgRNAs designed to CFTR gene: sgCFTR#1 - directly to the F508del mutation and sgCFTR#2 - 14 nucleotides 5’-upstream from the mutation. SgGFP designed to GFP gene was used as a control of the nuclease activity. PGEM-TA-CFTR plasmid with part of the CFTR gene with the F508del mutation co-transfected with the plasmid for CRISPR/Cas9 was used as template for sgCFTR#1 and #2; plasmid pEGFP-C1 was used as template for sgGFP. Experiments performed in HEK293T cell culture demonstrated, that sgCFTR#1 has the lowest efficiency among used sgRNAs - number of insertions/deletions (indels) by T7E1 assay was 6.37-20.82%. Expression level of sgCFTR#1 after transfection was lower than expression of sgGFP, which showed the greatest activity - up to 65% of indels. Addition of G-quadruplexes to sgCFTR#1 and sgGFP sequences with the aim to increase their stability led to decrease of expression and activity. Culturing transfected cells at lower temperature (24 hours at 37°C, then 48 hours at 30°C) resulted in two-fold decrease of sgCFTR#1 activity, but without affecting sgGFP activity. Thus, direct relationship between sgRNAs expression and their activity was confirmed in the study; however, sgCFTR#1 expression and its efficacy could not be increased. Further attempts to enhance sgCFTR#1 expression and its stabilization should be performed, or other Cas9 enzymes, which expand the ability to select sgRNA direct to F508del mutation can be potentially used.

In a 12-year-old female with early-onset epilepsy (remission), development delay, hyperkinesias and moderate lactic acidosis two independent rare disorders with overlapping phenotypes were detected by panel exome sequencing and confirmed by Sanger sequencing: autosomal dominant infantile epileptic encephalopathy type 42 (previously reported CACNA1A mutation p.Ala713Thr de novo ) and autosomal recessive mitochondrial respiratory chain complex I deficiency (compound heterozygosity for previously reported NDUFB3 mutations p.Trp22Arg and p.Cly70* in NDUFB3 gene). Clinical heterogeneity of NDUFB3 -related disease is discussed.

X-linked mental retardation, Cantagrel type ((MIM #300912; ORPHA:85277) is characterised by marked neonatal hypotonia, severely delayed developmental milestones, gastroesophageal reflux, stereotypic movements of the hands, esotropia and infantile autism. The article describes the case of mutation in the KIAA2022 gene in a 5-year-old girl with epilepsy, psychomotor, speech and intellectual development delay, behavioral disorders and autistic characters. Previously unknown heterozygous mutation in KIAA2022 gene , 3 exon (p.Asp451fs) was detected by targeted sequencing. Mutation was validated by the Sanger sequencing. The mutation was not found in parents of the child. Skewed X-inactivation was not detected in the study of CAG-repeat, AR gene, 1 exon in the proband. Mutations in the KIAA2022 gene can cause epileptic encephalopathy and intellectual disability in both boys and girls. It is important for genetic testing, medical management and genetic counseling.

Introduction: Congenital and hereditary diseases are largely represented by chromosomal pathology. In this regard, early diagnosis, treatment, rehabilitation, prevention of the origin and distribution of these diseases are of particular importance. Aim: Using a combination of cytogenetic methods, literature data and investigating several types of cells, to find out the genetic causes of disease in the patient. Materials and methods: Peripheral blood lymphocytes and skin fibroblasts were obtained from the proband. The study was performed using metaphase analysis, array comparative genomic hybridization (aCGH) on 8х60K microarrays (Agilent Technologies), real-time PCR and fluorescent in situ hybridization (FISH). Results: The patient with r(13) had several symptoms not associated with this chromosomal abnormality. In addition to the terminal del13q34, which caused the formation of r(13), trip3q12 was identified. In fibroblasts monosomy 13 was additionally detected during aCGH-analysis. Using FISH-analysis 47% and 50% of cell with monosomy 13 were detected among lymphocytes and fibroblasts, respectively. Conclusion: In the case of a chromosomal pathology a complex approach, involving the application of adequate research methods, analysis of several tissues in complex cases, work with literature and databases, and interaction of physicians and specialists of different profiles, will allow to explain the patient’s symptoms, provide an objective prognosis of the disease, and propose treatment.