Aim: description of a family case of ataxia-telangiectasia. Methods. Two brothers with atactic syndrome of unknown origin were examined. Molecular genetic study was performed by whole genome sequencing (LLC Evogen, Moscow). Results. A patient aged 1 year 9 months with symptoms of ataxia and an immunodeficiency state and his brother (6 years 9 months) underwent a clinical examination and molecular genetic testing. According to its results, 2 variants of the nucleotide sequence were found in the compound-heterozygous state of the ATM gene: in exon 40, leading to premature termination of p.Glu1978Ter protein synthesis (paternal origin) and in exon 58, leading to premature termination of p.Arg2849Ter protein synthesis (maternal origin) These variants are described in the compound heterozygous and homozygous state in patients with ataxia-telangiectasia. Conclusions. Establishing an accurate diagnosis for patients will allow timely determination of an observation plan that is interdisciplinary, as well as effectively carrying out preventive and rehabilitation measures. Medical genetic counseling of the family was carried out, the prognosis for the development of the disease for family members was clarified.

Hereditary sucrase-isomaltase deficiency is a type of hereditary disaccharidase intolerance. Homozygous forms of this pathology occur with a frequency of 0.02% in the population. The article describes a clinical case of this disease, identified by doctors in the Chelyabinsk region. A complete history, stages of the patient’s diagnosis, as well as the treatment used are presented.

Aim: description of an assisted reproductive technology (ART) program with preimplantation genetic testing (PGT) for spinocerebellar ataxia type 1 (SCA1) in combination with preimplantation chromosome analysis. Methods. Planning and implementation of PGT was performed for a couple (aged 30 and 31) from Sakha (Yakutia) at risk of SCA1. We have developed PGT system for monogenic diseases (PGT-M), which includes the analysis of the pathogenic variant of the ATXN1 gene (NM_000332.3(ATXN1):c.589_591CAG(36_38) (p.Gln208_His209ins(Gln)n) and polymorphic STR markers linked to the gene. For setup study DNA was isolated from the blood of the patient and partner, the patient’s sister and father, and a healthy unrelated donor. The study was performed by nested PCR with detection by fragment analysis. The developed system was validated on single cell samples. Ovulation stimulation and embryological procedures during the ART program were performed according to standard protocols, fertilization was performed by ICSI. Embryo biopsy was performed on the 5th day of development. Whole genome amplification (WGA) for trophectoderm samples was performed using multiple displacement amplification (MDA). WGA product was used for PGT-M according to the system developed at the setup stage, then for chromosomal microarray analysis of aneuploidy (PGT-A). The cryotransfer was performed taking into account the results of preimplantation testing. Results. The developed PGT-M system included analysis of the ATXN1 gene fragment carrying CAG repeats and analysis of 9 informative STR markers flanking the gene. Five mature oocytes were obtained in the ART program. Three embryos reached the blastocyst stage and were biopsied; WGA and PGT-M were performed for them. The mutant allele was well identified in two embryos. One of the three embryos was determined to be normal for the ATXN1 genotype and maternal STR haplotype. PGT-A was performed for one normal embryo, the result showed a normal chromosomal status. The embryo was transferred into the uterus. The results of hCG (human chorionic gonadotropin) confirmed the implantation of the embryo. Conclusions. Our clinical case demonstrates the successful implementation of the ART program with PGT-M for SCA1 in combination with the analysis of PGT-A.

Background. Proximal spinal muscular atrophy 5q is one of the most frequent autosomal recessive disorders worldwide, and is the most common cause of genetically related infant mortality. Globally, approximately 5% of patients are compound heterozygotes with a deletion in one copy of the SMN1 gene and a minor mutation in the other. However, there are only isolated studies on a small number of such cases in Russia. Aim: to study the minor variants spectrum of the SMN locus in proximal spinal muscular atrophy 5q patients. Patients and methods. The research was performed on DNA samples from 2164 unrelated patients referred with the diagnosis of proximal spinal muscular atrophy 5q. A control group consisted of 2973 unrelated individuals. SMN gene copy number was determined by quantitative MLPA. The search for minor mutations in the SMN1 gene was performed by Sanger sequencing. Results and conclusions. SMN1 and SMN2 gene copy number were examined in 2164 patients referred with a presumptive diagnosis of proximal spinal muscular atrophy 5q. The diagnosis of proximal spinal muscular atrophy 5q was confirmed in 1032 patients. Minor mutations in the compound heterozygous state with deletion of 7 and 8 exons were identified in 32 patients (1.5%), and among patients with 1 copy of SMN1 a minor mutation was detected in 43%. Variants (nonsense, missense, frameshift and splice site mutations) were identified in all exons of the SMN1 gene except 2a and 8. Recurrent mutations were detected in exon 6, two of which c.815A>G and c.821C>T occurred 6 and 7 times, respectively. Clinical type of proximal spinal muscular atrophy 5q was found to be associated with both mutation type and copy number of the SMN2 gene. The carrier frequency in the population according to our data is 1/35 people. The estimated carrier frequency of minor mutations in the population is 0.000224. Frequency of minor mutations in a population is 1/4464. The risk of having a child with 5q SMA in a family where the parents are silent carriers is 1/156240.

Hereditary connective tissue diseases are a large group of genetically and clinically heterogeneous diseases whose development is caused by mutations in the genes responsible for the synthesis of extracellular matrix proteins or involved in connective tissue morphogenesis. This article is devoted to the results of the search for the molecular genetic causes of the rare hereditary connective tissue diseases: Ehlers-Danlos syndrome, osteogenesis imperfecta and osteopetrosis. Diagnosis of these pathologies is difficult because of the similarity of symptoms and their clinical manifestations. The presented results will expand the understanding of the pathogenesis and will allow to optimize the diagnosis of these syndromes, to determine the tactics of medical and genetic counseling of burdened families by both clinical geneticists, orphan disease specialists, and general practitioners, family medicine specialists, and general practitioners.

90% of cases of congenital adrenal hyperplasia (CAH) are associated with changes in the CYP21A2 gene, of which about 20% are in various “chimeric” CYP21A1P/CYP21A2 genes. The problem of identifying “chimeric” genes and the relationship of their various types with the CAH clinical forms is still relevant today. The study is devoted to the analysis of the “chimeric” CH4 gene in the non-classical form of CAH. We used the methods of PCR-RFLP analysis and Sanger sequencing. DNA samples of 3 probands with a non-classical form of CAH and their parents were analyzed. The use of PCR-RFLP analysis with CYP21A2 gene-specific primers did not allow us to identify “chimeric” genes in the parents of the probands, but made it possible to assume their presence in the probands themselves. Whereas a complex analysis using all methods and pseudogene and gene-specific primers showed the presence in each of the 3 probands of the “chimeric” CH4 gene in the compound heterozygous state. In the first case, with the I173N variant, in the second, with i2 splice, and in the 3rd, with the 30 kb deletion of the CYP21A2 gene. For molecular genetic analysis in families with 21-hydroxylase deficiency, it is necessary to use both the analysis of major point mutations and methods that detect deletion-duplication events in the CYP21A2 gene. The presence of a “chimeric” CH4 gene with a break point between positions chr6:38038514 and chr6:32038938 (GRCh38) in a compound with other pathogenic variants leads to the nonclassical form of CAH.

The aim of this study was to evaluate effects of IL-17F gene polymorphisms, rs2397084 (substitution of glutamic acid (GAG) for glycine (GGG) at position 126; Glu126Gly) and rs763780 (substitution of histidine (CAT) for arginine (CGT) at position 161; His161Arg) on the cystic fibrosis (CF) patients mortality rate. The study involved 82 patients who were regularly examined (every 3-6 months) and received antibiotic therapy. The frequency of IL-17F (rs2397084) A and G alleles was 90.2% and 9.8%, respectively. The frequency of IL-17F (rs763780) A and G alleles was 92.1% and 7.9%, respectively. The patients were divided into two groups. Group 1 included individuals with common AA-AA genotype (n=55). Group 2 included carriers of one or more minor G alleles (n=27). The patients of two groups did not differ in age, sex, or number of F508del mutation carriers. At the same time, there were 14 dead patients in group 1, whereas all patients from group 2 were alive. Thus, survival in groups 1 and 2 was 74.5% and 100%, respectively (p=0.0035, Fisher’s exact test). Analysis of the Kaplan-Meier curves showed that the overall survival of patients in the group 1 was significantly lower than the corresponding parameter in the group 2 (p=0.0026, Log-Rank Test). Repeated microbiological studies of sputum samples revealed a significant predominance of patients with chronic colonization of the Burkholderia cepacia complex (Bcc) in this group: 22 vs 1 patient in group 2 (χ2 = 11.82, p = 0.0006; OR 17.33 [95% CI 2.19-137.21]). Thus, increased mortality rate in CF patients with common AA-AA genotype is associated with a high incidence of chronic Bcc infection. Further investigations with increased number of participants are needed to estimate more accurately the true risks of Bcc infection and poor outcomes in CF patients with different IL-17F gene polymorphisms.

Primary ciliary dyskinesia (PCD) is a rare and poorly studied disease, the phenotype of which is found with some common residual respiratory tracts that are difficult to diagnose. Thus, NGS techniques are becoming crucial in the diagnosis of this disease. At least 46 genes encoding various parts of the ultrastructure of the respiratory tract epithelial cilia and similar structures are involved in the pathogenesis of primary ciliary dyskinesia (PCD). Our research is aimed at finding the genetic cause of the disease and improving diagnostic efficiency in a group of patients with PCD. Bioinformatic analysis of whole exome sequencing data was performed for 19 probands from unrelated families under the age of 18 with a clinically established diagnosis of PCD. According to the data obtained, 14 (74%) of the 19 probands had genetic variants in the genes responsible for the formation of the PCD phenotype. Thus, an application of the complex approaches including methods of clinical genetics and molecular biology can improve the primary ciliary dyskinesia diagnostic efficiency.

Phenylketonuria (PKU) is a common, autosomal recessive hereditary disorder of phenylalanine hydroxylase caused by pathogenic PAH gene variants. After routine genetic analysis methods were applied, approximately 2% of PKU patients were not diagnosed. In this study, the search of nucleotide replacements were performed in noncoding and regulatory regions of PAH gen via NGS.The variant c.706+521G>C was found deep in intron 6, which in silico predicted the effect on splicing of the PAH gene. Thereby this study supplements current knowledge of PAH genotypes and show that deep intronic analysis of PAH can genetically diagnose PKU.

In most patients with male male infertility, it's cause remains unknown. Various chromosomal abnormalities and pathogenic variants in genes controlling spermatogenesis and male fertility are involved in the etiology of azoospermia and oligozoospermia. This study is aimed to improving the effectiveness of identifying the genetic causes of male infertility. For this purpose, a sample of 200 patients with idiopathic azoospermia or severe oligozoospermia was formed and examined. A comprehensive clinical and genetic examination was performed using both conventional methods (clinical and genetic, standard cytogenetic examination, analysis of the Y chromosome microdeletions at the AZF- locus and the CFTR gene), and genomic methods - massive parallel sequencing (MPS). Using “standard” diagnostic methods, genetic causes of infertility were found in 92 (46%) patients. Whole-exome sequencing (WES) revealed variants in 29 genes involved in the control of male fertility in 18 (42.9%) of 42 patients in whom genetic factors of infertility were not detected, according to the “standard” genetic examination, of which variants in 8 genes in 7 (16.6%) patients are presumably causative and require further functional or segregation studies. Comprehensive genetic examination, including genomic methods, makes it possible to increase the effectiveness of the diagnosis of genetically determined forms of infertility.

RPE65-associated retinopathies have received tremendous attention due to successful gene therapy. The current study is aimed at assessing the frequency of RPE65-dependent forms of hereditary retinal degeneration in the Russian Federation, as well as characterizing changes in the RPE65 gene in Russian patients. Of 189 unrelated patients with a referral diagnosis of RP or LCA, 11 patients with IRD with biallelic pathogenic variants in the RPE65 gene were identified, which accounted for 5.8% in the study sample. The most frequent mutation, c.370C>T (p.Arg124*), was found on five chromosomes. Two previously undescribed variants c.1024T>C (p.Tyr342His), c.1340T>C (p.Leu447Pro), classified as probably pathogenic, were identified. The significant contribution of RPE65-dependent forms of IRD to the Russian Federation and the possibility of gene therapy for these patients, as well as the absence of major mutations and hot exons in the RPE65 gene, proves the need for molecular genetic diagnosis of patients with various forms of IRD using the targeted next-generation sequencing (NGS) method.

Significant clinical and genetic heterogeneity of hereditary cataracts significantly complicates their early DNA diagnosis and determines the relevance of studying the epidemiology of the disease, population features of the spectrum and frequencies of mutations in responsible genes, clinical and genetic correlations. Mutations in the connexin 50 (GJA8) gene are common causes of hereditary, mainly autosomal dominant, congenital cataracts. In a patient with congenital bilateral zonular cataract in the GJA8 gene two nucleotide substitutions were identified in the compound heterozygous state - the previously undescribed missense variant C.143A>G (p.Glu48Gly) and the known nucleotide variant C.741T>G (p.Ile24Met). Based on clinical genealogical and molecular genetic analysis, the sporadic nature of the disease in the family was established. The newly identified variant of p.Glu48Gly is presumably a pathogenic mutation showing a recessive character.

Congenital merosin-deficient muscular dystrophy is an autosomal recessive form of muscular dystrophy characterized by muscle weakness that manifests itself at birth or in the first 6 months of life. The cause of this disease is a deficiency or complete absence of the merosin protein encoded by the LAMA2 gene. To date, there is no pathogenetic therapy for this disease, but various approaches for the treatment of MDCA1 are being actively developed around the world, and it is quite possible that an affordable therapy will exist in the near future. Therefore, today it is important to know the existing range of pathogenic variants characteristic of the Russian population, the most appropriate diagnostic methods and differential diagnosis with other neuromuscular diseases.

All PCR-based sequencing methods have a risk of allelic dropout (ADO) phenomenon, which leads to selective allele amplification during PCR process and may reduce the diagnostic yield of genetic testing. To identify the cases of ADO we compared BAM and VCF files with Sanger sequencing chromatograms. To reveal the causes of ADO, primer binding sites using the gnomAD database were analysed. All amplicons with suspected ADO cases were re-sequenced using the alternative oligoprimers pairs. We have identified 8 cases of ADO both in NGS sequences of targeted genes panels and direct Sanger sequencing. The fact of selective allele amplification was confirmed in all cases by re-sequencing using an alternative pair of primers. Most cases of ADO (6 cases, 75%) were caused by common or rare single nucleotide genetic variants at the annealing sites of oligoprimers, and in two cases (25%) ADO was presumably mediated by the presence of six- or more nucleotides indels in the amplicons studied. In addition, in some amplicons we found the “underrepresentation” of SNVs in NGS reads or the presence of the SNV only in the reads of one amplicon out of two. Given cases of proven and potentially ADO, we suppose that design of oligoprimers without registration of ADO phenomenon may affect the amplification efficiency up to 0,85% of amplicons.

Background. Multiple hereditary exostoses (MHE) or multiple osteochondromas (MO) (OMIM 133700, OMIM 133701) is a genetically heterogeneous disease manifested by generalized forms of skeletal damage with multiple progressive bone and joint deformities. The disease has an autosomal dominant type of inheritance, and the proportion of familial cases is exceedingly high. The article analyzes gene-phenotypic associations in a group of patients with multiple hereditary exostoses. Aim: to predict the development of clinical forms of MHE to improve the quality of life of patients. Methods. The results of clinical examination of 85 patients with MHE from 41 families with the results of molecular genetic studies were included in the study. The association of exostoses localization with clinical forms of MHE was analyzed by logistic regression with odds ratio calculation. Prognostic mathematical models were constructed. Congruence tables with the calculation of Fisher’s exact criterion were analyzed. Statistical analysis was performed using the application program package IBM SPSS Statistics 23.0. Results. According to the results of statistical analysis, it was found that clinical manifestations (exostoses localizations) in MHE depended on a specific mutation rather than on the gene (EXT1 or EXT2) in general. Individual combinations of exostoses localization were also shown to influence the development of clinical forms of the disease. Conclusion. The presence of stable gene-phenotypic associations makes it possible to predict the further clinical course of MHE in Yakut patients from the time of molecular genetic diagnosis.