The adenomas in Cushing’s disease frequently exhibit mutations in exon 14, within a binding motif for the regulatory protein 14-3-3 located between the catalytic domain (DUB), responsible for ubiquitin hydrolysis, and the WW-like domain that mediates autoinhibition, resulting in constantly active USP8. The exact molecular mechanism of deubiquitinase activity disruption in Cushing’s disease remains unclear. To address this, Sanger sequencing of USP8 was performed to identify mutations in corticotropinomas. These mutations were subjected to computational screening, followed by molecular dynamics simulations to assess the structural alterations that might change the biological activity of USP8. Eight different variants of the USP8 gene were identified both within and outside the “hotspot” region. Six of these had previously been reported in Cushing’s disease, while two were detected for the first time in our patients with CD. One of the two new variants, initially classified as benign during screening, was found in the neighboring SH3 binding motif at a distance of 20 amino acids. This variant demonstrated pathogenicity patterns similar to those of known pathogenic variants. All USP8 variants identified in our patients caused conformational changes in the USP8 protein in a similar manner. The identified mutations, despite differences in annotation results—including evolutionary conservation assessments, automated predictor data, and variations in localization within exon 14—exhibit similar patterns of protein conformational change. This suggests a pathogenic effect that contributes to the development of CD.

The Dicer protein is an indispensable player in such fundamental cell pathways as miRNA biogenesis and regulation of protein expression in a cell. Most recently, both germline and somatic mutations in DICER1 have been identified in diverse types of cancers, which suggests Dicer mutations can lead to cancer progression. In addition to well-known hotspot mutations in RNAase III domains, DICER1 is characterized by a wide spectrum of variants in all the functional domains; most are of uncertain significance and unstated clinical effects. Moreover, various new somatic DICER1 mutations continuously appear in cancer genome sequencing. The latest contemporary methods of variant effect prediction utilize machine learning algorithms on bulk data, yielding suboptimal correlation with biological data. Consequently, such analysis should be conducted based on the functional and structural characteristics of each protein, using a well-grounded targeted dataset rather than relying on large amounts of unsupervised data. Domains are the functional and evolutionary units of a protein; the analysis of the whole protein should be based on separate and independent examinations of each domain by their evolutionary reconstruction. Dicer represents a hallmark example of a multidomain protein, and we confirmed the phylogenetic multidomain approach being beneficial for the clinical effect prediction of Dicer variants. Because Dicer was suggested to have a putative role in hematological malignancies, we examined variants of DICER1 occurring outside the well-known hotspots of the RNase III domain in this type of cancer using phylogenetic reconstruction of individual domain history. Examined substitutions might disrupt the Dicer function, which was demonstrated by molecular dynamic simulation, where distinct structural alterations were observed for each mutation. Our approach can be utilized to study other multidomain proteins and to improve clinical effect evaluation.

Untranslated gene regions (UTRs) play an important role in controlling gene expression. 3′-UTRs are primarily targeted by microRNA (miRNA) molecules that form complex gene regulatory networks. Cancer genomes are replete with non-coding mutations, many of which are connected to changes in tumor gene expression that accompany the development of cancer and are associated with resistance to therapy. Therefore, variants that occurred in 3′-UTR under cancer progression should be analysed to predict their phenotypic effect on gene expression, e.g., by evaluating their impact on miRNA target sites. Here, we analyze 3′-UTR variants in DICER1 and DROSHA genes in the context of myelodysplastic syndrome (MDS) development. The key features of this analysis include an assessment of both “canonical” and “non-canonical” types of mRNA-miRNA binding and tissue-specific profiling of miRNA interactions with wild-type and mutated genes. As a result, we obtained a list of DICER1 and DROSHA variants likely altering the miRNA sites and, therefore, potentially leading to the observed tissue-specific gene downregulation. All identified variants have low population frequency consistent with their potential association with pathology progression.

Osteopetrosis is a group of rare inheritable disorders of the skeleton characterized by increased bone density. The disease is remarkably heterogeneous in clinical presentation and often misdiagnosed. Therefore, genetic testing and molecular pathogenicity analysis are essential for precise diagnosis and new targets for preventive pharmacotherapy. Mutations in the CLCN7 gene give rise to the complete spectrum of osteopetrosis phenotypes and are responsible for about 75% of cases of autosomal dominant osteopetrosis. In this study, we report the identification of a novel variant in the CLCN7 gene in a patient diagnosed with osteopetrosis and provide evidence for its significance (likely deleterious) based on extensive comparative genomics, protein sequence and structure analysis. A set of automated bioinformatics tools used to predict consequences of this variant identified it as deleterious or pathogenic. Structure analysis revealed that the variant is located at the same “hot spot” as the most common CLCN7 mutations causing osteopetrosis. Deep phylogenetic reconstruction showed that not only Leu614Arg, but any non-aliphatic substitutions in this position are evolutionarily intolerant, further supporting the deleterious nature of the variant. The present study provides further evidence that reconstructing a precise evolutionary history of a gene helps in predicting phenotypical consequences of variants of uncertain significance.

Humanin (HN) is a mitochondrial peptide that is encoded by the MT-RNR2 gene and can be composed of either 21 or 24 amino acids (aa), depending on whether it is expressed in the mitochondria or cytosol, respectively. It has cytoprotective, anti-inflammatory, and antioxidative properties that may involve a systemic normalization of stress-response homeostasis in many cell types. Moreover, HN analogs, such as S14G-humanin, have been synthesized and found to have strong neuroprotective effects. However, we still lack knowledge on the evolution of the MT-RNR2 gene and its cytosol-expressed products in different organisms. Using phylogenetic analysis, we have uncovered the scarcity of nuclear HN sequences in evolution and shown that true mitochondrial HN homologs are not likely to be found in non-vertebrates. Also, analyzing the diversity of HNs we have discovered that S14G substitution is presented among avian HN sequences while 21-aa HN is highly specific to mammals.