It is estimated that up to one-third of all variants causing inherited diseases affect splicing; however, their deleterious effects and roles in disease pathogenesis are often not fully characterized. Given their prevalence and the development of various antisense-based splice-modulating approaches, pathogenic splicing variants have become an important object of genomic medicine. To improve the accuracy of variant interpretation in public mutation repositories, we applied the minigene splicing assay to study the effects of 24 variants that were predicted to affect normal splicing in the genes associated with propionic acidemia (PA)—PCCA and PCCB. As a result, 13 variants (including one missense and two synonymous variants) demonstrated a significant alteration of splicing with the predicted deleterious effect at the protein level and were characterized as spliceogenic loss-of-function variants. The analysis of the available data for the studied variants and application of the American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP) guidelines allowed us to precisely classify five of the variants and change the pathogenic status of nine. Using the example of the PA genes, we demonstrated the utility of the minigene splicing assay in the fast and effective assessment of the spliceogenic effect for identified variants and highlight the necessity of their standardized classification.

Introduction. Over the past two years, there has been an increase in measles morbidity in Russia and other countries. In order to assess the heterogeneity of clinically significant strains of Measles morbillivirus to reveal the sources of infection and transmission routes strains, a molecular genetic study thus becomes an urgent task. In this paper, a genetic identification of clinical strains of measles virus detected in 2023–2024 is compared with global variants as well as Russian strains detected in previous years.Materials and methods. Forty measles virus genome sequences isolated from nasopharyngeal swab samples obtained in Moscow and Novosibirsk in 2023–2024 were included in the study. The data were then compared with strains collected in Russia in May 2023 and deposited at the Central Research Institute of Epidemiology, as well as strains collected in 2020 and 2021 in Moscow.Results. The nucleotide sequences of studied Measles morbillivirus strains were categorized into different phylogenetic groups within genotype D8. For samples of genotype B3 collected in 2020 and 2023, a comparative analysis was performed to identify the region of origin. Phylogenetic analysis of Russian and foreign variants of measles virus suggests that strains currently circulating in Russia may be a variety of strains that had previously circulated in other countries and independently spread to Russia in 2023. After analyzing the most frequent nucleotide substitutions in various measles virus genes, the most variable genes were identified to provide a basis for the extension of phylogenetic analysis.Conclusions. The proposed approach to molecular genetic testing of complete and partial genome sequences of clinical isolate of measles virus detected in 2023–2024 in Moscow and Novosibirsk made it possible to identify strain subgroups that differ in origin. The comparison of the Measles morbillivirus strains sequenced in the present research with global sequences allowed us to detect similar sequences identified both in 2023 and in previous years in various countries of the world. The analysis of epidemiologically significant strains of Measles morbillivirus shows that N gene can be used to reliably determine the main genotype; however, this approach is not sufficient for studying the transmission pathways of the virus.

The HGD gene encodes homogentisate 1,2-dioxygenase. A deficiency of this enzyme causes alkaptonuria (AKU; OMIM 203500), a monogenic autosomal recessive metabolic disorder. The global incidence of alkaptonuria is estimated at 1 in 250,000 to 1,000,000 live births. A large number of pathogenic nucleotide variants disrupt pre-mRNA splicing, leading to hereditary diseases. Many potentially splice-disruptive variants, including those in coding regions, remain uncharacterized. This lack of data makes clinical interpretation more difficult and can complicate diagnosis. We systematically analyzed 27 HGD variants predicted to affect splicing. Candidate variants from public databases (ClinVar, HGDdatabase) and our patient cohort were prioritized using in silico splicing predictions and evaluated with a minigene splicing assay in HEK293T cells. Based on the obtained functional analysis data, the variants were reclassified according to ACMG/AMP guidelines. In total, 13 variants changed their classification (9 were upgraded and 4 were downgraded), while 5 variants retained their pathogenicity class after analysis. Ten missense/nonsense variants were not reclassified, as no significant splicing disruption was detected. These findings improve the pathogenicity assessment of HGD variants, support more accurate diagnosis, and lay the foundation for future therapeutic strategies targeting splicing defects in AKU.