Svetlana S. Deryabina

In recent years, genetic testing for the diagnosis of hereditary forms of cancer has increasingly shifted to NGS technologies as a more sensitive and informative method. Objective. To find genetic variants in target regions of genes associated with hereditary types of breast and ovarian cancer by NGS in the biological material of patients with negative results on frequent mutations in the BRCA1/2 genes. Material and methods. The study included DNA samples extracted from tumor tissue and peripheral blood lymphocytes of 21 patient of the Sverdlovsk Regional Oncological Dispensary with breast cancer and negative results of searching pathogenic variants in BRCA1/2 genes by real-time PCR. Sequencing was performed on the MiSeq platform using the Solo-test ABC plus panel. Results. Using targeted NGS sequencing, 13 rare variants were identified in DNA samples from 11 women: seven germline (6 with established pathogenic significance and 1 VUS) and six somatic mutations (4 with activating action and 2 with unclear clinical significance). In four patients, rare BRCA1/BRCA2 mutations not included in the list of common Russian variants were found to be the cause of the disease, four women carried somatic alterations in PIK3CA, ATM and CHEK2, and three others had two variants in their DNA at the same time. Conclusion. The research substantiates the need for introducing multigenic-targeted sequencing into Russian clinical practice of breast cancer research.

Screening for carriage of hereditary diseases in most cases is the only way to establish the degree of risk of genetic pathology. Currently, neither in Russia nor in any other country in the world there is a definite list of genes and diseases associated with them, for which carriers should be tested, as well as normative documents for its implementation. The purpose of this literature review was to identify the factors influencing the attitude of both representatives of the medical community and the general population to the idea of mass introduction of genetic testing for carriage of hereditary diseases in persons of active reproductive age. Mainly publications for the last decade are analyzed, as well as international testing and tactics in detecting carriage of hereditary disease. Ethical and legal aspects of obtaining and using genetic information are highlighted. Further study and improvement of the regulatory and legal framework of genetic testing, taking into account the achievements of modern genetics, the emergence of new diagnostic technologies, moral, ethical and religious aspects of this problem are required.

Introduction. The rate of infertile couples comprises 17–24 % and tends to increase, with more of them turning to assisted reproductive technologies (ART). In case a required result is achieved, future parents-to-be prepare for birth of a healthy child in the family, but each couple may face a birth of offspring with severe genetic disorders including spinal muscular atrophy (SMA).Aim: to access a carriage rate for SMN1 gene exon 7 deletion associated with SMA and SMN2 gene copy number in married couples planning childbirth using in vitro fertilization (IVF).Materials and Methods. There were enrolled 170 couples (340 subjects) suffering from infertility and referred for IVF in the Sverdlovsk region (SR) of Russia. The search for deletions/duplications in the SMN1 and SMN2 genes was carried out by quantitatively analyzing number of gene copies using the SALSA MLPA Probemix P460 commercial kit (MRC-Holland, the Netherlands). The Hardy–Weinberg ratio was used to calculate estimated rate of homozygous carriers with SMN1 deletions in the next generation.Results. Among 340 patients, a deletion of exon 7 in the SMN1 gene (one copy out of two) was found in 9 individuals (3 males and 6 females) unrelated to marital relations. Thus, only 9 out of 340 examined subjects carry such gene mutation associated with SMA, with total rate of carriage comprised 2.65 % (1/38). Given the number of IVF procedures performed in SR, it may be assumed that the probability for birth of a sick child in such couple comprises at least 1:6410. A number of patients had SMN1 gene duplications – 9 (5.29 %) males and 4 (2.35 %) females baring 3 copies of the SMN1 gene. In addition, the majority of study participants (54 %) turned out to have 3 copies of the SMN2 gene.Conclusion. The rate of SMA carriage in married couples planning a pregnancy with aid of ART corresponds to the general population reaching 1:38 level. It is believed necessary that all couples entering the IVF program should be examined for carriage of SMN1 gene mutations to assess SMA risk in offspring.

Sverdlovsk Oblast has become one of the first Russia regions to participate in a pilot project of neonatal screening for the detection of severe forms of primary immunodeficiency (PID) in newborns yet back in 2021, prior to the nationwide rollout of the Expanded Screening Program. The purpose of this research was to evaluate preliminary the effectiveness of identifying children with severe combined immunodeficiencies as well as other life-threatening forms of lymphopenia at the stage of possible impact on the course and outcome of the disease. Materials and methods used: quantitative determination of TREC/KREC in dry blood spots was carried out in 5044 children born in the region in July-Sept. 2021. The testing was performed using the domestic “IMMUNO-BIT” kit by “ABV-TEST,” LTD (Skolkovo Innovation Center, Mozhaysky District, Moscow, Russia). Cutoffs suggested in the instructions were used: 500 copies per 105 leukocytes for TREC and 300 copies per 105 leukocytes for KREC. Results: the risk group included 157 newborns (68 girls and 89 boys) with low TREC and/or KREC values (3.1%), no children were found with a complete absence of markers. After testing the re-requested samples, 104 of them were determined to be false positive, and 53 families were invited for an immunologist’s appointment. 28 children received in-depth examination with the advisory. At the time of appointment, the condition of most of them was assessed as clinically stable, however, according to the results of laboratory and instrumental diagnostic examinations, the presence of immunological abnormalities and developmental features was demonstrated: changes in the thymus (50%), neutropenia (32%), lymphopenia and thrombocytopenia (28%), hypogammaglobulinemia (84%), congenital malformations (30%). Conclusion: for the correct understanding of the phenomenon of reduced TREC/KREC values (but not zero values) in some newborns and predicting outcomes, it is necessary to monitor the health status of these patients, which can be carried out through the formation of a dynamic observation group, which is impossible without active identification of such children through wide screening program.

Inborn errors of immunity can be detected by evaluating circular DNA (cDNA) fragments of T- and B-cell receptors (TREC and KREC) resulting from the receptor gene rearrangement in T and B cells. Maturation and activation of the fetal immune system is known to proceed gradually according to the gestational age, which highlights the importance of the immune status in premature infants at different gestational ages. In this article, we evaluated TREC and KREC levels in infants of various gestational ages by real-time PCR with taking into account the newborns weight and sex. The 95% confidence intervals for TREC and KREC levels (expressed in the number of cDNA copies per 105 cells) were established for different gestational groups. The importance of studying immune system development in newborns is informed by the discovered dependence of the level of naive markers on the gestational stage in the early neonatal period.

Chromosomal pathology is one of the most common causes of congenital malformations. The CATCH-22 symptom complex is most often associated with a microdeletion of chromosome 22, upon detection of which it is customary to diagnose DiGeorge syndrome, a known primary immunodeficiency or syndrome of innate errors of immunity. According to our data on the frequency of occurrence among all chromosomal abnormalities, DiGeorge’s syndrome takes second place in the Sverdlovsk region after Down’s syndrome, but its diagnosis is not simple due to varying severity of clinical manifestations, as well as different forms of the chromosome 22 defects. Along with several typical variants of 22q11 microdeletions, there duplications of critical regions are also reported, accompanied by immunodeficiency and other symptoms of CATCH-22. The effectiveness of diagnosing chromosomal abnormalities both in pre- and postnatal period largely depends on the grouping criteria of the patients with suspected chromosomal abnormalities, and on the methods used to identify hereditary pathology. In our study, we analyzed and compared the results of studies of 23 patients with various rearrangements of the 22q11.2 region, which were observed by a geneticist and clinical immunologist. The paper presents data on the polymorphism of phenotypes associated with rearrangements of the 22q11.2 region with an analysis of pathomorphological manifestations depending on the type of structural anomaly, i.e, del22q11.2, or dup22q11.2. The results of analysis demonstrate importance of different diagnostic options for laboratory studies of microdeletion and microduplication syndromes associated with immune-dependent pathology. We also compared the results of molecular genetic diagnostics and phenotypic manifestations in deletions and duplications of the 22q11.2 region. To identify the rearrangements of 22q11.2 region, two different methods were used – Prenatal BoBs and multiplex ligase-dependent probes’ amplification (MLPA). In particular, the both methods were used in the same patient to verify diagnosis, thus enabling to show differences in their efficiency. It was concluded that 22q11.2 deletion syndrome exhibits wide heterogeneity in phenotypic traits: neurological and immunological manifestations, anomalies in musculoskeletal development and internal organs, skull deformities and facial dysmorphia. Each clinical case was unique, requiring careful analysis of clinical manifestations. It is necessary to have a wide range of laboratory options for molecular genetic verification of the diagnosis.

Hennekam lymphangiectasia-lymphedema syndrome has been linked to single-nucleotide polymorphisms in the CCBE1 (collagen and calcium-binding EGF domains 1) gene. Several bioinformatics methods were used to find the most dangerous nsSNPs that could affect CCBE1 structure and function. Using state-of-the-art in silico tools, this study examined the most pathogenic nonsynonymous single-nucleotide polymorphisms (nsSNPs) that disrupt the CCBE1 protein and extracellular matrix remodeling and migration. Our results indicate that seven nsSNPs, rs115982879, rs149792489, rs374941368, rs121908254, rs149531418, rs121908251, and rs372499913, are deleterious in the CCBE1 gene, four (G330E, C102S, C174R, and G107D) of which are the highly deleterious, two of them (G330E and G107D) have never been seen reported in the context of Hennekam syndrome. Twelve missense SNPs, rs199902030, rs267605221, rs37517418, rs80008675, rs116596858, rs116675104, rs121908252, rs147974432, rs147681552, rs192224843, rs139059968, and rs148498685, are found to revert into stop codons. Structural homology-based methods and sequence homology-based tools revealed that 8.8% of the nsSNPs are pathogenic. SIFT, PolyPhen2, M-CAP, CADD, FATHMM-MKL, DANN, PANTHER, Mutation Taster, LRT, and SNAP2 had a significant score for identifying deleterious nsSNPs. The importance of rs374941368 and rs200149541 in the prediction of post-translation changes was highlighted because it impacts a possible phosphorylation site. Gene-gene interactions revealed CCBE1’s association with other genes, showing its role in a number of pathways and coexpressions. The top 16 deleterious nsSNPs found in this research should be investigated further in the future while researching diseases caused CCBE1 gene specifically HS. The FT web server predicted amino acid residues involved in the ligand-binding site of the CCBE1 protein, and two of the substitutions (R167W and T153N) were found to be involved. These highly deleterious nsSNPs can be used as marker pathogenic variants in the mutational diagnosis of the HS syndrome, and this research also offers potential insights that will aid in the development of precision medicines. CCBE1 proteins from Hennekam syndrome patients should be tested in animal models for this purpose.

Around 20,000 children with heart defects are born annually in the Russian Federation, among which around 5,000 children are manifested by its critical course that requires surgical correction within the first days after birth. Although timely pre- and postnatal diagnostics of congenital heart pathology allows to provide proper therapeutic and surgical assistance for such patients, it is hard to minimize severe complications at any stage of the child’s treatment. Quite often, post-surgery patients are unable to cope with serious complications caused by infectious diseases of the respiratory tract, endocardium and meninges. A special place is taken by cases of severe postoperative period: long-term non-healing postoperative wounds and septic disorders leading to patient death. Here we put forward an idea that congenital heart disease may serve as a marker of primary immunodeficiency characterized by lowered parameters of cellular and humoral immunity. Current retrospective study included 29 children died within the first year of life, whose medical records demonstrated confirmed congenital heart disease and manifested signs of immune-dependent pathology. There were analyzed data regarding pregnancy course in order to identify factors contributing to early diagnostics of immune-related pathology associated with CHD. The analysis of existing problems affecting proper diagnostics and treatment of children with congenital heart disease coupled to “covert” PID form was performed. It was noted that among twenty described syndromes manifested with congenital heart disease, seven are currently referred to the group of primary immunodeficiencies. Types of congenital heart defects in the study group ranged from disorders of the valve apparatus to the non-functional opening between heart chambers. Signs of immune-related pathology included decreased thymus size revealed by ultrasound examination as well as lowered value of genetic markers TREC and KREC, retrospectively obtained from neonatal dry blood spots. Finally, we name a key element for successful differential diagnostics of PID with congenital heart disease – awareness of various medical workers about such pathology for developing algorithms for their interaction with regard to management of such comorbid patients.

В ходе подготовительного этапа работы по внедрению генетического тестирования на тяжелый комбинированный иммунодефицит в программу неонатального скрининга в Свердловской области проведено исследование по количественному определению маркеров Т- и В-клеточного неогенеза (TREC и KREC соответственно) в крови условно здоровых новорожденных. Материалом исследования служили архивные образцы сухих пятен крови, собранных на тест-бланки для рутинного неонатального скрининга у 26 девочек и 26 мальчиков, родившихся доношенными и не имевших тяжелых заболеваний на первом году жизни. Дополнительно исследовано влияние гестационного возраста плода на количество данных маркеров (TREC и KREC) у недоношенных новорожденных разного срока гестации. Обследованы образцы крови 55 недоношенных детей, родившихся преждевременно (23-36 недель гестации). Показано, что уровни TREC и KREC последовательно возрастали с увеличением срока гестации, однако динамика данных изменений концентрации маркеров оказалась различной. В связи с этим рекомендуемый срок взятия образца крови для скрининга на ТКИН полностью соответствует срокам взятия образцов, принятым для большинства наследственных скринируемых заболеваний. Исключение составляет получение результата с полным отсутствием TREC или KREC в образце крови новорожденного любой степени недоношенности (при валидном количестве копий контрольного гена), что должно служить сигналом для незамедлительного назначения этому ребенку консультации врача-иммунолога и проведения углубленного иммунологического обследования, поскольку это может явиться первым прогностическим признаком фатального заболевания. Для получения корректных пороговых уровней TREC/KREC необходимы дополнительные исследования на большей выборке новорожденных (1000-5000) с последующей валидацией полученных референсных границ в исследованиях с включением больных с разными формами первичных иммунодефицитов.

Массовое обследование новорожденных в России на пять наследственных заболеваний не отвечает требованиям, предъявляемым мировым сообществом к программе неонатального скрининга. Успехи в развитии лабораторных диагностических технологий, активное внедрение в медицинскую практику достижений генетики и молекулярной биологии дают возможность пересмотра списка нозологий, входящих в национальную программу неонатального скрининга путем замены заболевания либо включения новых нозологий. В статье обсуждается возможность включения в программу скрининга новорожденных в России генетического тестирования на тяжелую комбинированную иммунную недостаточность. Обсуждаются результаты ретроспективного исследования маркеров наивных Т и В лимфоцитов — TREC и KREC — в группе детей с иммунозависимой патологией, развившейся на первом году жизни.