👤 Kirill Alexandrov

Genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. Analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereas FGF3, FGF4, FGF19 or CCND1 amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)-approved drugs. In conclusion, we identified risk factor-specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy. Show less

Hypertrophic cardiomyopathy is a common genetic cardiac disease. Prevention and early diagnosis of this disease are very important. Because of the large number of causative genes and the high rate of mutations involved in the pathogenesis of this disease, traditional methods of early diagnosis are ineffective. We developed a custom AmpliSeq panel for NGS sequencing of the coding sequences of ACTC1, MYBPC3, MYH7, MYL2, MYL3, TNNI3, TNNT2, TPM1, and CASQ2. A genetic analysis of student cohorts (with and without cardiomyopathy risk in their medical histories) and patients with cardiomyopathies was performed. For the statistical and bioinformatics analysis, Polyphen2, SIFT, SnpSift and PLINK software were used. To select genetic markers in the patients with cardiomyopathy and in the students of the high risk group, four additive models were applied. Our AmpliSeq custom panel allowed us to efficiently explore targeted sequences. Based on the score analysis, we detected three substitutions in the MYBPC3 and CASQ2 genes and six combinations between loci in the MYBPC3, MYH7 and CASQ2 genes that were responsible for cardiomyopathy risk in our cohorts. We also detected substitutions in the TNNT2 gene that can be considered as protective against cardiomyopathy. We used NGS with AmpliSeq libraries and Ion PGM sequencing to develop improved predictive information for patients at risk of cardiomyopathy. Show less

Bisphosphonate drugs such as zoledronic acid (ZOL), used for the treatment of common bone disorders, target the skeleton and inhibit bone resorption by preventing the prenylation of small GTPases in bone-destroying osteoclasts. Increasing evidence indicates that bisphosphonates also have pleiotropic effects outside the skeleton, most likely via cells of the monocyte/macrophage lineage exposed to nanomolar circulating drug concentrations. However, no effects of such low concentrations of ZOL have been reported using existing approaches. We have optimized a highly sensitive in vitro prenylation assay utilizing recombinant geranylgeranyltransferases to enable the detection of subtle effects of ZOL on the prenylation of Rab- and Rho-family GTPases. Using this assay, we found for the first time that concentrations of ZOL as low as 10nM caused inhibition of Rab prenylation in J774 macrophages following prolonged cell culture. By combining the assay with quantitative mass spectrometry we identified an accumulation of 18 different unprenylated Rab proteins in J774 cells after nanomolar ZOL treatment, with a >7-fold increase in the unprenylated form of Rab proteins associated with the endophagosome pathway (Rab1, Rab5, Rab6, Rab7, Rab11, Rab14 and Rab21). Finally, we also detected a clear effect of subcutaneous ZOL administration in vivo on the prenylation of Rab1A, Rab5B, Rab7A and Rab14 in mouse peritoneal macrophages, confirming that systemic treatment with bisphosphonate drug can inhibit prenylation in myeloid cells in vivo outside the skeleton. These observations begin a new era in defining the precise pharmacological actions of bisphosphonate drugs on the prenylation of small GTPases in vivo. Show less