A specific mutation commonly found in Parkinson’s patients has been shown to enhance the expression of α-synuclein (SNCA), a key gene involved in the pathogenesis of Parkinson’s disease. These findings were published in Nature, in the study “Parkinson-associated risk variant in distal enhancer of α-synuclein modulates target gene expression.”
Genome-wide association studies (GWAS), which examine genetic variants in individuals to identify disease-associated variants, have identified several genetic variants that are associated with complex diseases. However, how specific risk variants functionally contribute to the underlying pathogenesis of these diseases remains largely unknown.
Parkinson’s disease, the second most common chronic progressive neurodegenerative disease, is often caused by mutations in the coding sequence or multiplications of the SNCA gene. However, single nucleotide polymorphisms (SNP) — mutations that modify one nucleotide in the genetic sequence, and the mutation type most commonly detected in GWAS — affect the binding of transcription factors and can also affect gene expression.
“The generation of patient-derived human induced pluripotent stem (iPS) cells, which carry all pathogenic genetic alterations, is attractive for the study of diseases. However, significant biological heterogeneity due to differences in genetic background, variation in human iPS cell isolation, and in vitro differentiation present a serious limitation for identifying a disease relevant phenotype in the culture dish,” the authors wrote. “This is particularly relevant for sporadic diseases likely displaying only subtle in vitro phenotypes.”
GWAS studies have identified the SNCA genomic region (locus) as the one associated with higher risk of sporadic development of Parkinson’s. The research team, from The Whitehead Institute and the Massachusetts Institute of Technology in Massachusetts, now aimed to identify disease-associated SNPs in the enhancers — regions of the DNA that can bind to transcription factors to activate gene transcription — of the SNCA locus.
Results revealed that the top seven risk variants were localized to two distal enhancer elements, one in the intron 4 and other in the 3’UTR, regions of non-coding DNA. One particular variant from the intron-4 enhancer (re356168 A/G), where an adenine (A) was replaced by a guanine (G), showed higher differences in predicted transcription factor binding.
To dissect the effect of this genetic variant on SNCA gene expression, researchers used the clustered regularly interspaced short palindromic repeats (CRIPR)/Cas9 genome editing tool to insert the rs356168 SNP or to delete the enhancer in vitro in only one chromosome, leaving the other chromosome unchanged to act as an internal control. Their results showed that SNCA expression was increased when cells were carrying the G allele at rs356168, but not with the A allele or when the enhancer was not present. Analysis of 86 post-mortem samples of Parkinson’s disease supported the functional effect of rs356168, as carriers of this risk allele showed a significant increase in total SNCA levels.
Differential transcription factor binding is considered to be a major mediator of the effects on gene regulation observed upon specific mutations in distal enhancers. Consistently, the researchers identified two transcriptional factors expressed in the brain, NKX6-1 and EMX2, that bind preferentially to the protective lower SNCA-expressing A allele of rs356168, suggesting that these transcription factors bind to the enhancer and repress its activity, modulating SNCA expression.
In their study, the authors described “an alternative experimental approach to identify functional risk variants based on three recent innovations in genetics and molecular biology: (i) the prioritization of GWAS-identified risk variants in regulatory elements such as distal enhancers annotated based on genome-scale epigenetic data; (ii) the generation of genetically controlled isogenic pluripotent stem cell lines in which specific disease-associated genetic variants are the sole modified experimental variable using efficient gene-editing technologies such as the CRISPR/Cas9 system; and (iii) the analysis of cis-acting effects of candidate variants on allele-specific gene expression through deletion or exchange of disease-associated regulatory elements.
“This approach eliminates the effect of system inherent variability such as in vitro differentiation and results in an internally controlled experimental system, which allows robust and reproducible identification of cis-acting sequence-specific effects on gene regulation,” the researchers concluded.