At present, with the advanced affordable genetic testing, the rate of discovering amyotrophic lateral sclerosis (ALS)-related genes rapidly increases. These genetic findings provide new insights into therapies that target genetic subset of ALS. However, the research on the genetic and environmental causes of ALS is still in the early stage. In this chapter, we review the current understanding of ALS-related genes and summarize the worldwide ALS distribution feature by the frequency of occurrence in different regions. We summarize the advances in genetic testing and counseling for ALS. Based on the increase in genetic testing, we believe that the ALS patients and families would be benefited from our studies in the near future.
- genetic frequency features
- genetic testing
- genetic counseling
- genetic therapy
Amyotrophic lateral sclerosis (ALS) is an adult onset and generally fatal neurodegenerative disease characterized by progressive weakness and atrophy of voluntary skeletal muscles due to dysfunction and death of upper and lower motor neurons. Onset typically occurs between 60 and 69 years of age, with wide-range severity. About 90% of cases are sporadic amyotrophic lateral sclerosis (SALS), while familial amyotrophic lateral sclerosis (FALS) accounts for the remaining 10% cases . The pathogenesis of ALS remains obscure, but genetic mutations have been accounted for several impaired cellular and molecular mechanisms and, thus, provide clues for potential therapeutic strategies.
The superoxide dismutase 1 (
In most cases, FALS is inherited in the dominant pattern and the penetrance is associated with age. It has been observed that the differences of age onset and disease progression within and between ALS families are significant. In addition, some ALS is recessive inherited, such as
Some ALS caused by specific genetic mutations exhibit unique clinical characteristics. For example, ALS associated with
Recently, progress in gene discovery and technology has both complicated and empowered the process of genetic testing options, which may help neurological clinicians and ALS patients to understand the pathogenesis of ALS, and then provide genetic counseling for family members, allow accurate risk assessment, and open the door for genotype-specific treatments. As the genetic basis of the remainder of FALS, and potentially SALS, is unraveled, genetic testing and counseling will become increasingly vital and should be incorporated into the routine management of ALS [4, 5, 6].
2. Recent advances in ALS gene map
The superoxide dismutase 1 (
The FUS RNA-binding protein (
CCNF protein is a kind of kithe cell cyclin, involved in the regular of cell cycle transitions by activating cyclin-dependent protein kinases. Furthermore, it is the substrate recognition component of the Skp1-cullin-F-box E3 ubiquitin ligase complex. The neurons of the over-expressed mutant CCNF showed an increase in protein-labeled proteins, including TDP43. It indicates that mutated-CCNF protein interfere the proteasome degradation pathway by using traversing protein abnormalities to mark all proteins or inhibiting transferring transprotein-labeled proteins to proteasome complexes. This finding suggests that the
The TANK-binding kinase 1 (
Some patients also reported with extrapyramidal, ataxia, or psychosis. Neuropathological examination of central nervous system (CNS) tissues from patients with
2.8 Pathogenesis of ALS-related genes
The cellular processes, including RNA processing, protein degradation pathways, ubiquitin-proteasome system (UPS), autophagy, and so on, are all reported related to ALS pathogenesis [1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22]. Sorted by the various processes, we summarize the causative genes and genes might increase susceptibility of ALS which impact physiological activities mentioned above (Figure 1).
3. GWAS on ALS
Recently, the studies on ALS have shown the development trend of the blowout with the technology improving, however, no longer limited by technical condition, the number of newly discovered ALS-related gene did not meet expectation. It indicates a shift in the genetic pathway that multiple genetic variants and environmental factors may interact simultaneously to increase ALS susceptibility. On the basis of the fact that sporadic form apparently accounts for high rate of ALS and the hypothesis that ALS may not appear to be familial in a conventional Mendelian manner, a new research method, genome-wide association study (GWAS), is applied to the research of ALS.
3.1 SNPs and GWAS
The International HapMap Project, which began in 2002, mapped the single-nucleotide polymorphisms (SNPs) haplotypes of the human genome from four major populations in the world and promoted the development of GWAS. At the same time, the rapid development of high-density and high-throughput genotyping technology, which can detect hundreds of thousands of SNPs in a single reaction, makes it possible to systematically screen mutations associated with complex diseases throughout the genome. Unlike previous candidate gene studies, GWAS does not need to build any assumptions based on disease pathophysiology prior to the study and can relatively be an unbiased screen for almost all common mutations in the genome. At present, some risk factors of complex diseases, such as age-related macular degeneration, diabetes mellitus, breast cancer, and so on, have been initially identified by GWAS.
3.2 ALS GWAS boot
Schymiek et al. firstly reported GWAS in SALS in February 2007 . A total of 276 patients and 271 controls with white American ancestry were recruited in this research. They used a chip to detect 555,352 SNPs and found 34 of the most relevant SNPs by association analysis. For negative results, they elucidated that SALS might contain a group of diseases with similar clinical manifestations like FALS, each of which is determined by different mutation sites, and that different diseases and mutation sites may interfere with GWAS’s shooting out of susceptible genes. Among the 34 SNPs, there was an overexpression of genes associated with cytoskeletal actin regulation. For example, the
Recently, a series of ALS GWAS studies have been published and found several potential risk genes [29, 30, 31, 32]. However, the results of these studies are different with the same ideas and methods. The biological process of some candidate genes is unclear, and the evidence needs to be supplemented.
4. Genetic testing
Gene testing helps ALS patients and families enhance their understanding on the condition and information requested in genotype-specific treatments. Although ALS patients desire access to genetic testing, genetic advances have been slow to reach the clinical care of the ALS patient. In recent years, the landscape of genetic testing and genetic counseling for ALS has been rapidly transformed with the identification of novel genes and the advent of next-generation sequencing technology.
4.1 Genetic testing options
As with all clinical testing, genetic risk assessment, including family history and pedigree analysis, and pretest counseling, helping patients anticipate the possible impact of genetic testing on themselves and their family members, are necessary for patients before genetic testing.
Currently available genetic testing options for ALS include Sanger sequencing for traditional simple mutations, assays for the
4.2 Post-test counseling
Regarding the positive result, specific mutation, genotype-phenotype correlations, family history, and inheritance pattern should be thoroughly analyzed. Meanwhile, implications and risks for family members, including offspring and siblings, and theories about why the disease occurs also should be reviewed and addressed. For the reported definite pathogenic mutations, clinicians should provide information and hope about the potential genetic therapies in the future.
4.3 Presymptomatic testing
To increase certainty, make health or lifestyle choices, and make decisions about family planning, the presymptomatic testing could be conducted. According to the guidelines for presymptomatic genetic testing in other neurodegenerative diseases such as Huntington disease and Alzheimer disease, ALS should be tailored as following: pretest genetic counseling, baseline neurologic and cognitive assessment, psychological evaluation, in-person disclosure, presence of support person, and posttest genetic counseling. Most of important, presymptomatic testing should be offered to adult first-degree relatives of ALS patients with established mutations after written informed consent obtained .
5. Gene therapy
With the exception of riluzole, an anti-glutamatergic agent which was shown to prolong survival for 2–3 months by blocking the presynaptic release of glutamate, and edaravone, an antioxidant which was shown to decrease the rate of patient immobility, no effective treatment is currently available for ALS that can stop or reverse the disease progression.
Gene therapy is a promising therapeutic approach for ALS since it can be used to deliver “gene drugs,” encoding for blocking the novel gene expression, antiapoptotic proteins, and for neurotrophic factors, to the motor neurons crossing the blood-brain barrier specifically to prevent further motor neuron degeneration and to preserve the function of remaining motor neurons.
Here, we are to illustrate some examples of each therapeutic strategy for describing the present status and advance of gene therapy treatment.
The neurotoxicity of mutant SOD1 is related to the dose of the toxic protein through multiple pathological mechanisms. A potential therapeutic approach to SOD1-related ALS is to block the expression of the toxic SOD1 that could cause motor neuron degeneration . This therapy option which is more worthy of attention is that it possibly avoids and decreases potential influence in downstream pathological cascades. Recently, the studies mostly focus on antisense oligonucleotides and RNA interference which are both to block gene expression through enhancing the degradation of RNA.
5.1.1 Antisense oligonucleotides in models and human
In animal models of
The first clinical trial of antisense oligonucleotide treatment in human beings had favorable safety outcomes, and now the clinical trial to assess the safety, tolerability, and pharmacokinetics of a second generation SOD1 antisense oligonucleotide is in progress (Clinical Trials.Gov, NCT02623699) .
5.1.2 Short hairpin RNA (shRNA) treatment in mutant SOD1 ALS models
According to literature, in
While, in a recent study reported, SOD1 expression in the motor cortex of P70
5.1.3 miRNA treatment in mutant SOD1 ALS models and healthy nonhuman primates
In a study, scientists reported a new method that systemically delivered drug based on an artificial microRNA. In the
Similar therapeutic approach targeting C9orf72 for ALS is also in development. The toxicity of mutated C9orf72 is imparted by the formation of nucleolar RNA foci that sequester important RNA-binding proteins and by the generation of toxic dipeptide repeat (DPR) proteins.
The C9orf72 hexanucleotide repeat expansion (HRE) of GGGGCC DNA and RNA enables the formation of complex structures including G-quadruplexes. Because the Guanosine-rich DNA and RNA sequences are prone to formation of G-quadruplexes, a stable four-stranded structure present within ribosomal DNA sequences, transcription start sites, the promoter and untranslated regions of mRNA, human telomeric DNA sequences. It may play an important role in various cellular processes such as telomere maintenance, ribosome biogenesis, gene replication, transcription, and translation. Therefore, both C9orf72 HRE DNA and RNA may contribute to the pathogenesis of ALS/FTD disease through a mechanism associated with their structure polymorphism. Presently, based on the above mechanism, two strategies including antisense-mediated interventions and small-molecule-based approach have been developed to interfere with neurodegenerative diseases associated with G-quadruplexes .
5.2.1 Antisense oligonucleotides in mutant C9orf72 ALS models
A recent study reported that the RNA foci and DPR proteins were reduced significantly in mutant C9orf72 ALS mice by a single-dose injection of antisense oligonucleotides to reduce C9orf72 RNA repeats, and after 6 months of treatment, the motor function was also preserved .
5.2.2 Small-molecule ligands targeting the G-quadruplex structure in mutant C9orf72 cells
The small-molecule ligands, such as porphyrin, acridine, pentacridium, telomestatin, naphthalene diamide, and bisquinolium, directly target and bind to the G-quadruplex structure and selectively modulated the function of the G-quadruplex. For example, TMPyP4, a cationic porphyrin, can bind and disrupt the secondary structures of C9orf72 HRE and even damage its interactions with hnRNPA1 and ASF/SF2 proteins .
Similarly, some studies also showed that three small-molecule ligands can bind G-quadruplex and decreased RNA foci and RNA translation in both cultured cells and patient-derived neurons .
TBK1 is a key regulatory molecule upstream of OPTN, SQSTM1/p62, and IRF3 in the autophagy and neuroinflammatory pathways that are implicated in ALS. Manipulation of TBK1 might potentially compensate for defects caused by other ALS-associated proteins in these pathways—for example, VCP and UBQLN2. NEK1 and C21orf2 are known to interact at the protein level and, in addition to TUBA4A, PFN1, NEFH, and PRPH, they represent the building blocks of the cellular scaffold. Administration of small molecules that enhance cytoskeletal integrity could represent a viable therapy for stopping progression or reversing the disease course in patients with these mutations [4, 5, 6].
Nearly a decade ago, the only way to test ALS-related gene was SOD1 sequencing, whereas clinicians now have a wide availability of testing options already. Whole-exome sequencing is current standard in most related searches. However, the factors such as high rate of incomplete penetrance in ALS, few large pedigrees, and short survival of patients lead to the discovery of ALS-related genes worse than expected and the identification of susceptible mutations limited.
Although many known ALS-related genes’ structural characteristics and roles have been discovered, which have highlighted critical processes, pathways, and intracellular localizations of dysregulation, there are still many reported variants with uncertain significance. Further functional studies are needed to clarify the pathogenesis of these genes. Till now, more people believe that ALS is the result of interaction between multiple genes that each increases the susceptibility of the disease, but does not initiate the pathogenesis alone. So, we need to do more research on oligogenic ALS cases. Most importantly, with the improvement of understanding of ALS genetics, we will have more opportunities to develop meaningful therapies.