Spectrums of Amyotrophic Lateral Sclerosis. Группа авторов
effect.
NONCODING VARIATION
Investigation of variants associated with ALS has been mainly limited to coding regions. As genomic research progresses, more is known about noncoding regions, allowing a larger scope of inquiry.
Regulatory and Intronic Variants
Although most variants currently associated with ALS are in coding regions of genes, some discoveries of noncoding regions have been made. Variants in the untranslated regions of transcripts are generally regulatory, affecting the level and localization of transcripts [90], while intronic variants might affect splicing and nuclear export of transcripts [91]. While a rare variant is not causal simply because it is in a known ALS gene, at least some noncoding variants in these genes are likely implicated in disease risk. As the association of noncoding variants with ALS has not often been replicated or expressly studied, these variants may represent a significant proportion of missing heritability, and genetically explained cases could be higher than currently reported. While it is rare that a single variant has a strong effect on ALS risk, ALS cases appear to have a higher burden of rare variants in the 3’UTR region of known ALS genes [86]. For example, variants in the 3’UTR of FUS gave elevated FUS expression and higher risk for ALS [92] and were observed in 1.2% of ALS cases in a study cohort [92]. Several ALS‐associated genes encode for RNA binding proteins [17, 21, 36], which could interact with these untranslated regions of ALS genes. As an example, TDP‐43 binding to intronic and 3’UTR regions of its RNA targets is one of its normal functions, and without proper binding, TDP‐43 has a significantly higher tendency to form cytoplasmic aggregates [31].
Epigenetics
Epigenetic modifications, such as post‐translational histone modifications and DNA methylation, are a mostly unexplored area of ALS genetic etiology. These modifications tend to increase or decrease the expression of genes, rather than alter the function of the gene directly. Because these marks can be impermanent, modulation of these modifications might lead to new therapeutics. However, because post‐transcriptional and post‐translational modifications tend to be ubiquitous and varied in usage across the genome and cell types, it will require a targeted and informed approach to manipulate these modifications. Two examples of epigenetic study are those of CpG islands in and around the C9orf72 HRE and genome‐wide histone acetylation.
Penetrance of the C9orf72 HRE is strongly age‐dependent, ranging from 60% at 60 years old to over 90% at 80 years old [8]. If cellular modulation could lower the expression of the C9orf72 HRE, perhaps this would explain the large variance in age at symptom onset for HRE carriers. Since promoter methylation allows for the restricted expression of the downstream gene, several studies have shown a link between methylation of a promoter sequence upstream of the C9orf72 gene and lowered transcription of the HRE [93–95]. The promoter is only hypermethylated in cis with the HRE [93], and non‐carriers show little to no methylation [94].
The C9orf72 HRE is a repeat of (GGGGCC)n and is therefore composed of several potential CpG sites. Whether this is of consequence to ALS genetic etiology is still in question. Early reports did not observe methylation of the HRE itself [93], but later experiments demonstrated size‐dependent methylation of the HRE [96, 97]. Since the C9orf72 HRE shows somatic instability when expanded [43], the methylation status of the HRE itself could be of interest in terms of cell‐specific effects and time/age‐dependent aspects. Indeed, both repeat dipeptide proteins and RNA foci are reduced in cell models of methylated HRE [97]. Whether this differential methylation is modifiable is another important consideration, since methylation patterns and age are closely correlated [98]; whether aging changes methylation patterns and therefore gene expression, or whether changes in methylation patterns result in increased aging, is unknown (for a thorough review on genomic considerations of epigenetic changes and aging, refer to Pal and Tyler [99]).
Another level of epigenetic regulation of gene expression is that of histone modifications or post‐translational modifications. Observations of an upregulated microRNA (miR‐206) in a transgenic hSOD1 p.G93A mouse model led to speculation that its target mRNA, histone deacetylase 4 (HDAC4), could implicate chromatin remodeling in ALS [100]. Further, in ALS patients with more rapid symptom progression, HDAC4 expression was found to be upregulated, and muscle reinnervation was lessened [101]. If miR206 targets and lowers the expression of the muscle‐specific HDAC4, and HDAC4 decreases muscle reinnervation, it would follow that the protective effects of miR206 could be reproduced by inhibiting the action of HDAC4. Broadly inhibiting Class II HDAC proteins was found not to be effective in increasing ALS survival in hemizygous hSOD1 p.G93A mice, although glutamate receptor uptake was restored [102]. Inhibiting HDAC4 directly in mouse skeletal muscles through knockout led to worsened symptoms and disease severity [103]. Even though increased expression of HDAC4 correlates with ALS symptoms, its inhibition also leads to worsened symptoms [103, 104]. Unfortunately, although current research can identify modified pathways and responses to ALS cellular pathology, a clear picture of epigenetic regulation is not yet available for ALS, and further research will be needed before inhibitors can be used therapeutically [104].
CONCLUSIONS
Genetic research of ALS focuses on what causes the disease, but also on the biological implications of carrying associated variants. Many of the variants associated with ALS lead to protein misfolding or aggregation. However, it is uncertain whether misfolding and subsequent aggregation is a cause of disease or a hallmark of normal cellular response to abnormal proteins. As more biological pathways such as RNA metabolism, mitochondrial function and survival, nuclear‐cytoplasmic trafficking, and synaptic transmission are being implicated in ALS, a more complete schema will emerge. One variant may be important to ALS, but as so many ALS cases are without penetrant inherited variants that it is likely a larger‐scale consideration of the genome will lead to better understanding of the disease process.
ACKNOWLEDGMENTS
We thank Cal Liao, Cynthia Bourassa, Fulya Akçimen, and Zoe Schmilovich for reviewing the manuscript. JPR has received a doctoral student fellowship from the ALS Society of Canada and currently receives a Canadian Institutes of Health Research Frederick Banting and Charles Best Canada Graduate Scholarship (FRN 159279).
CONFLICT OF INTEREST
The authors declare no potential conflict of interest with respect to research, authorship, and/or publication of this manuscript.
COPYRIGHT AND PERMISSION STATEMENT
To the best of our knowledge, the materials included in this chapter do not violate copyright laws. All original sources have been appropriately acknowledged and/or referenced. Where relevant, appropriate permissions have been obtained from the original copyright holder(s).
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