Discovery and verification of functional single nucleotide polymorphisms in regulatory genomic regions: Current and developing technologies

Abstract

The most common form of genetic variation, single nucleotide polymorphisms or SNPs, can affect the way an individual responds to the environment and modify disease risk. Although most of the millions of SNPs have little or no effect on gene regulation and protein activity, there are many circumstances where base changes can have deleterious effects. Non-synonymous SNPs that result in amino acid changes in proteins have been studied because of their obvious impact on protein activity. It is well known that SNPs within regulatory regions of the genome can result in disregulation of gene transcription. However, the impact of SNPs located in putative regulatory regions, or rSNPs, is harder to predict for two primary reasons. First, the mechanistic roles of non-coding genomic sequence remain poorly defined. Second, experimental validation of the functional consequences of rSNPs is often slow and laborious. In this review, we summarize traditional and novel methodologies for candidate rSNPs selection, in particular in silico techniques that aid in candidate rSNP selection. Additionally we will discuss molecular biological techniques that assess the impact of rSNPs on binding of regulatory machinery, as well as functional consequences on transcription. Standard techniques such as EMSA and luciferase reporter constructs are still widely used to assess effects of rSNPs on binding and gene transcription; however, these protocols are often bottlenecks in the discovery process. Therefore, we highlight novel and developing high-throughput protocols that promise to aid in shortening the process of rSNP validation. Given the large amount of genomic information generated from a multitude of re-sequencing and genome-wide SNP array efforts, future focus should be to develop validation techniques that will allow greater understanding of the impact these polymorphisms have on human health and disease.

A critical assessment of the factors affecting reporter gene assays for promoter SNP function: a reassessment of −308 TNF polymorphism function using a novel integrated reporter system

“…Along with the gel shift assay, reporter gene analysis is used to establish a prima facie case for further, more detailed investigation of a particular SNP or indel that shows an association with a particular phenotype. 5,6 As outlined earlier, the contribution of any individual regulatory SNP to phenotypic variation is likely to be small. The transient reporter gene assay is not designed to reliably measure statistically meaningful differences of these magnitudes.…”

GH secretagogue receptor gene polymorphisms are associated with stature throughout childhood

“…Given that SNPs located within noncoding regions such as promoters, enhancers, and silencers may impact gene regulation (25), it is possible that rs482204 and rs562416, both located within the 3 0 -UTR outside putative functional domains, may act as regulatory SNPs or affect mRNA stability. Alternatively, they may be in LD with a functional variant.…”

SNPs, Linkage Disequilibrium and Transcriptional Factor Binding Sites Associated with Acute Mountain Sickness among Han Chinese at the Qinghai-Tibetan Plateau

“…SNPs which occur in the putative regulatory region of a gene where a single base change in the DNA sequence of a potential TFBS may affect the process of gene expression are drawing more attention [40][41][42] …”

“…In which cases the gene is no longer regulated by the original TF. Therefore, functional regulatory(r) SNPs in TFBS may result in differences in gene expression, phenotypes and susceptibility to environmental exposure [42]. Examples of rSNPs associated with disease susceptibility are numerous and several reviews have been published [42][43][44][45].…”