Management of Gene Promoter Mutations in Molecular Diagnostics

Abstract

BACKGROUND:Although promoter mutations are known to cause functionally important consequences for gene expression, promoter analysis is not a regular part of DNA diagnostics.CONTENT: This review covers different important aspects of promoter mutation analysis and includes a proposed model procedure for studying promoter mutations. Characterization of a promoter sequence variation includes a comprehensive study of the literature and databases of human mutations and transcription factors. Phylogenetic footprinting is also used to evaluate the putative importance of the promoter region of interest. This in silico analysis is, in general, followed by in vitro functional assays, of which transient and stable transfection assays are considered the gold-standard methods. Electrophoretic mobility shift and supershift assays are used to identify trans-acting proteins that putatively interact with the promoter region of interest. Finally, chromatin immunoprecipitation assays are essential to confirm in vivo binding of these proteins to the promoter.

Bioinformatic Analysis for the Validation of Novel Biomarkers for Cancer Diagnosis and Drug Sensitivity

“…As previously mentioned, acquisition of mutations in the promoter regions of the tumour suppressor genes can alter their expression (Vooght, K. et al 2009) and ultimately down-regulate the control of proliferation and cell death (Chial, 2008). The different types of mutation ultimately will impact on the outcome, with point mutations, insertions and deletions being common types of mutation that can have very dissimilar effects on the cell (Loewe, 2008).…”

P2‐030: Investigating the Role of Clu, Picalm, and Cr1 in Alzheimer's Disease

“…When subjected to electrophoresis, labelled DNA alone will show a different motility than DNA bound by TFs, and any differences in pattern between the sequences could indicate disruption of TFBS. Alternatively, the wild-type and variant sequences could be cloned upstream of a reporter gene in a promoter construct plasmid and be transfected in to a relevant cell line, with any differences in reporter gene expression indicative of differential activity (de Vooght et al 2009). …”

Gene promoter analysis in molecular diagnostics: do or don’t?

“…There is hardly any literature regarding the incidence of promoter mutations in relation to disease. However, the incidence of reported promoter mutations in genes seems to depend on the extensiveness by which genes have been studied in the past and the severity of disease caused by the mutation [3]. Examples of diseases caused by promoter mutations are b-thalassemia [4], Bernard-Soulier syndrome [5], pyruvate kinase deficiency [6], familial hyper cholesterol emia [101] and hemo philia [7] (for a review, see [8]).…”

“…The analysis of disease-causing promoter mutations typically consists of in silico analysis of the promoter mutation, functional promoter assays, such as transient transfection assays, and DNAtranscription factor binding assays, such as electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays [3]. Overall, promoter assays are complex and time-consuming, making specific demands on laboratory equipment (i.e., cloning and cell culturing facilities).…”

“…Therefore, we believe that routine laboratories should cooperate more with research groups involved in gene promoter research. This will work in both directions: routine laboratories can translate the findings of research groups into diagnostic tools, whereas groups involved in gene promoter research, depend on the identification of these mutations in patients, to improve knowledge on transcriptional regulation Editorial Gene promoter analysis in molecular diagnostics of the gene of interest and the role of disturbed transcriptional regulation in disease [3]. Improving the data on the in vivo and in vitro effects of promoter mutations associated with disease will hopefully lead to a proper assessment of the incidence of disease causing promoter mutations with respect to other known genetic causes of disease, free of most bias or prejudice.…”