Postzygotic somatic variants in cancer-associated genes underlie a range of overgrowth and vascular malformations syndromes. PIK3CA-related overgrowth spectrum (PROS) is one of the key diseases in this group, which encompasses more than 20 conditions marked by asymmetric overgrowth and vascular malformations. The PIK3CA gene encodes the catalytic subunit p110α of phosphatidylinositol- 3-kinase and is mutated in various types of cancer. The mutational events in PROS occur during embryonic development, resulting in a mosaic distribution of mutations. The diversity and severity of PROS clinical manifestations are determined by a combination of factors, including the degree of PI3K activation by specific variants, the type of affected tissue, the timing of mutation occurrence, and the influence of additional factors. The high phenotypic variability of PROS, its similarity to other overgrowth vascular syndromes, as well as the challenges of molecular genetic diagnosis of mosaic variants, complicate the diagnosis of PROS. In this work, we summarize the current knowledge about the molecular mechanisms of PROS, the involvement of the regulatory subunit in the pathogenesis of PROS, and discuss the diagnostic and therapeutic features of this condition.

Background. The vast majority of currently discovered missense variants have unknown clinical significance. In this regard, the classification of such variants is an urgent problem of medical genetics, since the inability to establish the clinical significance of a variant complicates the diagnosis of inherited diseases, as well as the development or application of existing therapeutic strategies. In this work, a new bioinformatics tool AlphaMissense was used to assess the efficiency of variant classification in the TCF4 gene.

Aim: prediction of the pathogenic effect of all possible missense variants in the TCF4 gene using the AlphaMissense tool based on machine learning, and evaluation of the ability to classify variants by this tool using ROC analysis.

Methods. The following were used to create and analyse the data discussed in this paper: Google Colab development environment, Python v3.10 programming language, Biopython library for working with biological sequences, scikit-learn library for ROC analysis. The TCF4 gene sequence contained in the NCBI database was used as a reference. 1241319 single nucleotide polymorphism (SNP) variants were generated, among which 6906 variants are in the coding sequence, of which 3747 were identified as missense variants. Annotation of the obtained data was performed according to ClinVar and AlphaMissense databases using the OpenCRAVAT tool. Of all the detected missense variants, 979 variants were scored by AlphaMissense, of which only 101 variants were reported in the ClinVar database.

Results. When comparing sensitivity (Se), specificity (Sp), ROC curve plots and area under the curve (AUC) values, there is a clear difference in the evaluation of SNP classification as likely pathogenic (AUC = 0.81, Se = 0.68, Sp = 0.78). It can be used as an additional criterion in screening of candidate variants for Pitt-Hopkins syndrome. In contrast, classifying variants as likely benign or ambiguous lacks sensitivity and specificity, and their AUC scores characterise them as models of medium quality. Therefore, the variants included in these groups require further reassessment by other tools.

Conclusions. The measured values make it evident that the AlphaMissense tool is best at identifying likely pathogenic variants. However, variants identified as likely benign or ambiguous should be considered questionable and should be tested with other tools. Variants obtained by artificial mutagenesis and assessed as likely pathogenic but not listed in databases may be useful in identifying previously unknown variants in the TCF4 gene and help in the diagnosis and development of therapies for associated diseases.

Mutational events in the genes of the epigenetic apparatus lead to changes in DNA methylation. Unique DNA methylation patterns (episignatures) are highly specific biomarkers of diseases and can be used for indirect diagnosis of chromatinopathies and for interpretation of variants in epigenes. In this work, we searched for the most significant CpG dinucleotides from the genome-wide episignature of Sotos syndrome and developed a classification model for indirect diagnosis of Sotos syndrome based on the identified episignature. This model is applicable as a second-line test (in the clinical picture of Sotos syndrome and the absence of a molecular genetic diagnosis). The presence of the episignature of Sotos syndrome establishes the diagnosis and prompts the search for the causative variant in the NSD1 gene.

Background. Determination of chromosomal imbalance has an important clinical and biological significance in cases of spontaneous abortion. It is necessary to establish the cause of non-pregnancy, to exclude hereditary factors, to provide further genetic counselling, and to understand the mechanisms of chromosomal anomalies. The gold standard for karyotype analysis is microscopic analysis of chromosomes after GTG staining. However, this method has limitations as it depends on the mitotic activity of the cells and the presence of well-visualised chromosomes. An alternative cytogenomic approach to detect chromosomal imbalance may be low-coverage and ultra-low coverage whole-genome sequencing, used to detect aneuploidies and large copy number variations.

Objective: to perform whole-genome sequencing with ultra-low coverage for molecular karyotyping of spontaneous and medical abortuses.

Methods. Uncultured material of extraembryonic mesoderm of spontaneous and induced abortions was used. Molecular karyotyping was performed using ultra-low coverage whole genome sequencing. The results were verified using fluorescence in situ hybridization (FISH) and real-time PCR.

Results. Among spontaneous abortuses, aneuploidies were detected in 25 out of 71 (35.2%) cases, among which autosomal trisomies were the most frequent abnormality (92%), while sex chromosome number abnormalities were detected in 8% of cases. Polyploidy was detected in 4 out of 71 cases (5.6%), giving a cumulative incidence of chromosomal abnormalities of 40.8%.

Conclusions. Determination of the efficacy and specificity of ultra-low coverage whole-genome sequencing for the detection of aneuploidy showed values of 100% in both measures on the control groups. Confirmation of karyotype abnormalities in spontaneous abortions by reference methods showed that ultra-low coverage whole-genome sequencing is effective in the diagnosis of aneuploidy in the absence of alternative methods.

It is known that the placenta of human embryos in the I trimester of pregnancy and fetuses in the II and III trimesters are enriched with CNVs. This property is assumed to be necessary for the normal functioning of placenta and successfull pregnancy. At the same time, pathogenic CNVs associated with known microdeletion and microduplication syndromes were detected in placentas of spontaneous abortions (SA). However, these data were obtained on DNA isolated from the mixture of placental cells, while placental tissues in I trimester can be divided into chorionic villi (CV) and extraembryonic mesoderm (EM), originating from different germ layers. The study of two tissues allows tracking the distribution of CNVs in the placenta, identifying de novo variants that arose after the separation of germ layers, and, in the presence of parental DNA, identifying inherited CNV. Here, for the first time, CV and EM (34 SA), as well as parental DNA (17 couples) were studied on Agilent 180K microarrays. CNVs were detected in one or both tissues in 21 (62%) SA. A total of 226 variations were identified, of which 126 (56%) were detected in one of the tissues (75 in CV (33%) and 51 in EM (23%)); at the same time, 100 CNVs (44%) were present in both tissues. In CV, the variants were found 1.5 times more often than in EM. The ratio of de novo and inherited CNVs was 3:1. Pathogenic and likely pathogenic variations were present either in both tissues or only in CV.

Imprinting disorders (ImpDis) are a consequence of violations of the mechanism of genomic imprinting, which regulates the expression of homologous alleles of genes of different parental origin in imprinted regions of chromosomes. A solitary ImpDis is characterized by a methylation anomaly within its specific imprinted region. Multilocus imprinting disturbances (MLID) are characterized by methylation abnormalities in several imprinted regions. Four classes of molecular anomalies have been established for ImpDis: deletions/duplications, uniparental disomies, epimutations, and mutations in imprinted genes. MLID are detected more often among patients with epimutations. The causes of MLID are pathogenic variants in genes encoding oocytic and zygotic factors of embryo development, such as NLRP2, NLRP5, NLRP7, KHDC3L, OOEP, PADI6, TLE6, UHRF1, ZFP57, ARID4A, ZAR1, ZNF445, TRIM28, involved in the life cycle of imprinting. The presence of causative variants in MLID-associated genes in the family of a patient with MLID significantly increases the risk of reproductive losses and the birth of children with ImpDis and MLID, as opposed to single-locus epimutation within one imprinted chromosomal region, which makes it important to follow-up on MLID among patients with single-locus epimutation. In the research, methylation-specific multiplex ligation-dependent probe amplification of regions 6q24.2; 7q32.2; 11p15.5; 14q32.2; 15q11.2; 19q13.43; 20q13.32 (MS-MLPA MLID) was applied to 209 DNA samples of patients both sexes. In a group of patients with previously confirmed ImpDis without CNVs (113 samples), previous results were confirmed in 113 cases, and MLID were additionally detected in 7 (6.2%) cases. The comparison of ImpDis’s spectrum between samples with previously confirmed ImpDis without CNVs and newly identified diagnosis shows an increase in the detection of the Temple syndrome from 1 (0.9%) case to 5 (15.2%) cases using МS-MLPA MLID. Among group of patients with previously unconfirmed diagnoses (25 samples), not only have we successfully confirmed diagnoses with molecular genetics in 6 (24.0%) cases, but also, we have identified methylation abnormalities different from the expected clinical syndromes in 2 (8.0%) cases, and MLID in 4 cases (16.0%). In the group of patients newly referred for the diagnosis (56 samples), clinical diagnoses were confirmed by molecular genetics in 17 (30.4%) cases, different molecular disorders were detected in 3 (5.4%) cases, MLID were detected in 1 case (1.8%). Based on the results of the assessment of the phenotype of patients with MLID, it is shown, that the phenotypic signs of MLID may coincide with the signs of known ImpDis, partially overlap with the signs of some known ImpDis and can be non-specific, which makes clinical diagnosis difficult. During the whole-genome analysis of patient with TNDM-MLID, previously undescribed homozygous variant NM_001109809.4.8(ZFP57): c.115delG (p.Val39fs) has been revealed and assessed as likely pathogenic (PVS1+PM2) according to ACMG criteria. Both parents are heterozygous carriers, which determines the risk of rebirth of a child with TNDM-MLID at 25%. Based on the results of the research, the algorithm for the diagnosis of ImpDis has been proposed, with MS-MLPA MLID as first-line test.

Klinefelter syndrome (KS) is the most common gonosomal aneuploidy caused by the presence of one, rarely 2-4 additional X chromosomes in the karyotype in male patients. The phenotypic variability of the disease may be associated with the influence of the cytogenetic variant of KS, as well as other genetic and epigenetic factors. A number of studies have established the influence of parental origin and inactivation of the X chromosome, as well as its genes and their variants, in particular the CAG polymorphic locus of the androgen receptor (AR) gene. However, the frequency of individual allelic variants and genotypes for this polymorphic locus have not been studied in KS patients. In this study, the CAG polymorphic locus of the AR gene was analyzed in 222 KS patients. The number of trinucleotide repeats was determined using the PCR-based AFPL (amplified fragment lengths polymorphism) method. The number of CAG repeats varied from 14 to 32, the average number was 22.3 ± 2.7. In the studied sample, 59 patients were homozygous, 163 patients were heterozygous at this locus. In both groups, the frequent alleles contained 20-25 CAG repeats, the median was 22. A statistically significant (p < 0.05) difference was found in the allelic frequency of the most common variant of CAGn, n=21 between homozygotes and heterozygotes. The revealed difference for CAGn polymorphic locus of the AR gene between homozygous and heterozygous KS patients can be explained by possible differences in the frequency of sex chromosomes non-disjunction in the germ cells of their parents, which requires further investigation.

DNA copy number variations (CNVs) are responsible for intellectual disability, autism spectrum disorders, attention deficit hyperactivity disorder, and other nervous system diseases. The same pathogenic CNVs can be inherited from healthy parents and are also found in intrauterine deceased human embryos, raising the question of incomplete penetrance and pleiotropic properties of some variations. In this paper, we report a case of a monogenic microduplication in the 2q23.1 region containing the MBD5 gene inherited from a healthy father, and consider the possible association of this microstructural chromosomal abnormality not only with the birth of an affected child but also with pregnancy loss.

Mucopolysaccharidosis type IIIC (MPS IIIC, Sanfilippo syndrome) is an inherited disease that is caused by pathogenic variants in the HGSNAT gene, which encodes the lysosomal enzyme alpha-glycosaminidase N-acetyltransferase. The characteristic phenotype is the primary indication for referral for biochemical diagnosis of accumulation diseases and subsequent molecular genetic study. The individual was observed to display delayed psychomotor development and behaviors consistent with autism spectrum disorder. Upon external examination, the subject displayed a few distinctive physical characteristics, including a coarse facial features, a dolichocephalic skull shape, stiff dark hair, thick eyebrows, a protruding lower lip, a short neck, hypertrichosis, a cylindrical chest, an umbilical hernia, long arms, and flat-valgus feet. The patient displayed a delayed psychomotor development and exhibited behaviors consistent with autism spectrum disorder. The biochemical diagnostic tests yielded results indicating an elevated total concentration of glycosaminoglycans. The results of one-dimensional electrophoresis of glycosaminoglycans demonstrated an elevated excretion of heparan sulfate in the urine. A molecular genetic study identified two pathogenic variants in the HGSNAT gene that were inherited from carrier parents. Based on the clinical data, results of biochemical and molecular genetic studies, and a comprehensive analysis, the diagnosis of ‘Mucopolysaccharidosis type IIIC’ was established. In conclusion, the case study illustrates the characteristics of MPS IIIC, including its typical phenotypic features and central nervous system involvement.