Laboratory of Molecular Genetics

& Pharmacogenomics-Toxicogenomics

Director: Dr. Sotiria Boukouvala, Associate Professor

 

Democritus University of Thrace, Department of Molecular Biology and Genetics

Building 10, University Campus, Alexandroupolis 68100, Greece.

Tel./Fax.: +30-25510-30632, email: sboukouv@mbg.duth.gr

More info: http://utopia.duth.gr/~sboukouv/Sotiria_Boukouvala_BIO.pdf and http://nat.mbg.duth.gr/

 

Scientific interests

Our main research interest is in pharmacogenomics and toxicogenomics, characterizing genes that modulate the effects of chemicals (drugs, carcinogens, toxic agents etc.) on living organisms. We undertake comparative genomic and functional investigations to unravel how genetic diversification and metabolic adaptability have enabled organisms to survive adverse chemical environments. Current projects investigate the family of genes for arylamine N-acetyltransferases (NATs). These are xenobiotic metabolizing enzymes with diverse functions in prokaryotic and eukaryotic microorganisms of medical and/or biotechnological relevance. In humans and animal models, the highly polymorphic NAT genes have been landmark examples of how genetic variability may determine individual susceptibility to chemical carcinogenesis and drug-induced toxicity.

More recently, our interest has shifted to the study of microbial secondary metabolism, as we found several bacterial NAT homologues to reside within biosynthetic gene clusters, where they encode enzymes that catalyze a range of “unexpected” biochemical reactions. We have explored the evolutionary relationships between those divergent NAT homologues, and we have traced back the evolutionary origins of the NAT family to transglutaminases and cysteine proteases.

The experimental approaches of the group are broad, ranging from in silico comparative genomic surveys to gene and gene cluster cloning, combined with functional investigations at the genetic, molecular and biochemical level, both in vivo (using microorganisms or cell lines) and in vitro (using recombinant proteins or by chemically analysing metabolites).

The group maintains a biotechnological orientation, building on previous experience of the PI in the industry. Current ventures include:

·       characterization (through Nanopore whole-genome analysis), genetic engineering, and heterologous expression of bacterial biosynthetic gene clusters, aiming to produce and chemically identify new secondary metabolites with potential pharmaceutical interest,

·       activation of biosynthetic gene clusters via the co-culture of bacterial strains, followed by chemical analysis of possible antimicrobial secondary metabolites,

·       genetic and structure-function investigation of fungal NAT enzymes that enhance the pathogenic fitness of endophytic fusarium infecting grains,

·       comparative investigation of bacterial tolerance and metabolic activity towards aromatic amines, exploring the role of NAT genes in the detoxification of those highly toxic byproducts of industrial activity, farming and everyday life.

Other research projects of the group focus on biomedicine and pharmacogenetics, including:

·       pharmacogenetic validation of human variability at the NAT locus (copy number variation and haplotype determination) via in silico analyses, digital PCR and Nanopore sequencing,

·       pharmacogenetic investigation of ADME genes in human populations,

·       study of the role of human NAT1 gene in estrogen receptor positive vs. negative breast tumours,

·       comparative investigation of NAT genes in non-human primates as pharmacological in vitro models for human NATs.

Previous projects have additionally included genetic investigation of an affected family by whole-exome trio analysis, a cross-border collaboration with Bulgaria on the molecular diagnosis of sexually transmitted viral infections, an industry-academia collaboration exploring nanotechnologies for targeted delivery of anti-cancer drugs, as well as pharmacogenomic investigations into the prediction of drug-resistance mutations in cancer pharmaceutical targets.

 

Expertise and infrastructure

The laboratory shares its infrastructure with the Laboratory of Human Genetics & Model Organisms and is fully equipped to support methodologies ranging from in silico genomic surveys to advanced genetic, molecular and biochemical applications. Current expertise includes high-throughput PCR, real-time PCR, digital PCR, cloning, protein expression and purification, western blot, various spectrophotometric, chemiluminescent and fluorescent assays, differential scanning fluorimetry, pulsed field gel electrophoresis, cell culture, microbiology, Nanopore sequencing technology, etc.

Available equipment includes a DNA 4x96 Engine Tetrad for PCR (MJ), a TECAN M1000 reader for colorimetric, chemiluminescent or fluorescent in vitro/cell-based high-throughput assays, a BioRad ChemiDoc XRS+ Imaging System for gels and chemiluminescent blots, a Nanodrop spectrophotometer (Thermo), a BioRad Experion Automated System for chip-based electrophoresis and qualitative/quantitative analysis of RNA/DNA and protein, various standard and specialized electrophoretic instruments (e.g. for PFGE, E-gels etc.), electroporator, sonicator, various incubators, a -80oC freezer etc.

The laboratory has three dedicated areas, one for PCR (with sterile hood), a second for microbiology (equipped with a BL2 hood, a shaking incubator and microscopes) and the third for cell culture (equipped with a BL2-Cytotoxic hood, an inverted microscope, a CO2 incubator and accessory instruments). Access to communal state-of-the-art facilities is also available on campus.

 

International Collaborations

The group has established international collaborations with the following institutions:

·       Department of Pharmacology, University of Oxford (UK), on functional investigations of NAT genes and their recombinant protein products.

·      Department of Microbiology, Eötvos Loránd University of Budapest (Hungary), on studies of NAT genes in bacteria.

·      Toxicology and Mycotoxin Research Unit, Agricultural Research Service, US Department of Agriculture (Athens, GA, USA), on studies of NAT genes in fungi.

·      University of Paris-Décartes and University of Paul Sabatier-Toulouse (France), on studies of NAT gene function and evolution in primates.

·      Department of Genetics and Evolution, University of Geneva (Switzerland), on population pharmacogenomic studies.

·      Department of Biomedical Sciences, University of Queensland (Brisbane, Australia), on studies into the evolutionary relationships between human NATs and other enzyme families.

·      Department of Pharmacology and Toxicology, University of Louisville (KY, USA), on studies of NAT pharmacogenetics and nomenclature.

·      Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, Oswaldo Cruz Foundation (Rio de Janeiro, Brazil), on population pharmacogenetic studies.

·      Pharmacogenetics Core Laboratory, Clinical Pharmacology & Medical Toxicology, Children’s Mercy Research Institute (Kansas City, MO, USA) on NAT pharmacogenetics and allelic nomenclature.

 

The PI also participates as appointed expert of NAT pharmacogenetics, in the following international initiatives and consortia:

Responsibilities include: assigning of official symbols to new NAT genes and alleles; recommending changes to the current nomenclature system for NAT genes; liaising with other gene nomenclature committees (e.g. HGNC) and with the NAT scientific community; collection, annotation and presentation of genomic data on the NAT website (http://nat.mbg.duth.gr/).

The aim of the panel is to catalogue allelic variation of NAT2 gene impacting drug metabolism, under a unifying nomenclature system for all pharmacogenes. Efforts are synchronized between PharmVar, the PharmGKB and CPIC. The panel holds monthly meetings to discuss progress and make decisions with regard to the transitioning of NAT2 nomenclature from the legacy to the new system (https://www.pharmvar.org/).

The initiative brought together stakeholders from the pharmacogenetics community (US/EU agencies, gene nomenclature committees, gene variant databases, professional societies and standards development organizations, industrial test developers, clinical and research laboratories, etc). The workgroup held regular conference calls to develop consensus recommendations for clinical and research laboratories to standardize pharmacogenetics nomenclature and test result reporting. The workgroup recommendations were published in 2016.