Causes of FTD: C9orf72 Gene Mutation

Hexanucleotide repeat expansion mutations in the chromosome 9 open reading frame 72 (C9orf72) are commonly found in frontotemporal dementia (FTD) patients, accounting for 25.9% of known FTD cases[1]. This mutation is characterized by duplications of GGGGCC sequences in the gene and is hypothesized to be one of the genetic causes of FTD[1][2]. Although correlations have been identified between FTD patients and C9orf72 mutation carrier, the function of the protein encoded by the C9orf72 gene as well as the mechanism by which the mutation causes FTD remains largely unknown. However, potential roles of the gene and disease mechanisms have been postulated. Further investigation into this gene is expected to provide insight into the disease and play an integral role in finding a cure to FTD.

Figure 1
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The C9orf72 gene. The hexanucleotide repeat region is shown above the arrow.[4]

Dementia Page = Dementia

1. C9orf72 gene

1.1 Background information on the C9orf72 gene

Chromosome 9 open reading frame 72 (C9orf72) is a gene which encodes an uncharacterized protein with unidentified function [1]. The protein encoded by the C9orf72 gene is thought to belong to a family that is known to exchange guanine nucleotides for Rab GTPases [3]. The C9orf72 sequence is also highly conserved across multiple species and likely plays an important role in the function of the organism [2]. The gene contains a GGGGCC hexanucleotide repeat, which exists upstream of the C9orf72 protein coding region and is located between two non-coding exons [5]. Depending on the length of the repeat, it is located in either the promoter or in an intragenic region of the C9orf72 gene [1]. The transcripts yielded from this sequence are found in various tissues throughout the body [2]).

1.2 Hexanucleotide repeat expansion mutation in C9orf72

Hexanucleotide repeat expansion mutation is defined as an expansion in the GGGGCC-repeat [1]. In the case of the C9orf72 gene, hexanucleotide repeat expansion occurs between the two non-coding exons, where the GGGGCC repeat sequence is located [1]. The number of repeats in an individual with no expansion mutation in the C9orf72 allele is thought to range between two to thirty repeats [1]. Carriers of C9orf72 repeat expansion mutations are somatically heterogenic and thus the number of repeats varies from a tissue to another [1]).
According to the paper published in 2014 by Pliner, Mann and Traynor, the origin of the hexanucleotide expansion in C9orf72 is hypothesized to be from a single event that took place in Finland in 500 A.D. [6].

1.3. C9orf72 gene mutation as the cause of Frontotemporal Dementia

According to recent papers, hexanucleotide repeat expansion mutation in the C9orf72 allele is thought to account for 25.9% of non-sporadic frontotemporal dementia (FTD) cases and 5.1% of the sporadic FTD cases, making it the most important genetic cause of FTD [1]. The expansion mutation in C9orf72 gene is found to be more common among FTD patients than other FTD-causing mutations such as microtubule associated protein tau (MAPT) mutations [2]. C9orf72 gene mutation and mutations in FTD-associated genes often co-occur, explaining the pleiotropic nature of the disease in C9orf72 mutant carriers [1]. Additionally, researchers postulate the instability and the expanding nature of the hexanucleotide repeat as the reason why FTD increases in severity and leads to earlier onset in successive generations [1]. Additional research must be conducted to uncover the mechanism by which the mutation in the C9orf72 gene leads to frontotemporal dementia.

2. Potential Disease Mechanisms

Figure 2
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A graphic representation of the
G-quadruplex structure[5]

The mechanism in which hexanucleotide repeat expansion in C9orf72 causes FTD remains largely unknown [1]. Two of the disease mechanisms that are proposed as the method in which the mutation causes FTD include: the loss-of-function disease mechanism due to a decreased level of C9orf72 transcript, and the RNA-protein sequestration caused by gain-of-function toxicity of mutant RNA [7].

2.1. RNA toxicity: Sequestering of repeat-binding proteins by mutant gene transcript

In C9orf72 mutant gene carriers, intracellular accumulation of the mutant transcripts leads to
a detrimental RNA foci formation in the nucleus and in the cytoplasm [2]. RNA foci formed in the expanded mutant carriers are toxic aggregates with high levels of stable G-quadruplex secondary structures [7]. This stable complex is created by expanded hexanucleotide repeats transcripts and sequesters RNA-binding proteins, altering downstream pathways [2]. It has been shown that RNA-binding proteins such as heterozygous nuclear ribonucleoprotein (hnRNP) and ASF/SF2 splicing regulator are sequestered and their functions are altered as a result of RNA foci binding [7]. In C9orf72 mutant gene carriers, RNA foci were detected in the ¼ of the cells in the frontal cortex and spinal cord, and this is suspected to be the cause of FTD in expansion carriers [2].

2.2 Reduced gene expression: Low C9orf72 mRNA level in mutant

Figure 3
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The diagram at the very top is an illustration of the C9orf72 locus.
The three diagrams underneath it are the three transcript variants
that are derived from alternative splicing.[2]

It has been discovered that the C9orf72 gene gives rise to at least 3 different C9orf72
transcripts via alternative splicing, which generate 2 different isoforms of the C9orf72 protein [2]. The transcript variants yield isoforms that vary in amino acid length, and it has been found out that the level of one of the alternatively spliced transcripts, which encodes for a 481 residue protein, is severely reduced in C9orf72 mutant carriers [1]. From this, loss-of-function pathomechanism has been suspected as the cause of FTD [1]. Researchers suspect the reduced transcript level to be due to either haploinsufficiency, which is the reduction of gene product from the mutant gene, or by a change in the alternative splicing pattern in expanded allele carriers [4]. It is hypothesized that the reduction in C9orf72 gene expression is the result of the low transcript levels, and that the expression change are due to the hypermethylation of CpG islands and interactions between hexanucleotide repeat expansion and the trimethylated histones [3]. The hypermethylation and the interaction of the repeat with the histones are presumed to increase the difficulty of accessing the C9orf72 coding region, thereby reducing the transcript level [3]. Currently, the direct effect of the reduction in transcript is unidentified; further investigation is required to elucidate the role of C9orf72 transcripts in FTD.

2.3 Reduced C9orf72 protein level in mutants

C9orf72 protein is found to be significantly reduced in the frontal cortex of individuals harboring the expanded allele, and this is thought to be one of the causes of FTD in C9orf72 mutant carriers [3]. Transcript levels were also found to be low in long repeat carriers, suggesting that this is the cause of decreased protein levels [3]. It has also been determined that in some cases, despite carrying C9orf72 gene with a non-pathogenic repeat length, the low level of C9ORF72 can lead to FTD [3].

3. Potential Cure for FTD

3.1. TMPyP4: Counteracts RNA toxicity

The accumulation of toxic RNA in expanded allele carriers leads to the formation of G-quadruplex structures called RNA foci, which sequester RNA-binding proteins in 25% of the cells in the spinal cord and the frontal cortex and lead to FTD [2]. In a recent paper published in 2014, Zamiri B, Reddy K, Macgregor RB Jr, and Pearson CE provide TMPyP4 as a potential solution to the RNA toxicity in C9orf72 mutant [7]. TMPyP4 is a catatonic porphyrin that can bind to the G-quadruplex and thereby ablates the detrimental sequestration of RNA-binding proteins [5]. The TMPyP4 binding causes G-quadruplexes to undergo a conformational change [7]. The distortion of the secondary structure of the stable G-quadruplex structure diminishes the ability of RNA foci to interact with proteins, thereby recovering the RNA-binding proteins that have been sequestered by the RNA foci [7].

3.2 Developing antisense oligonucleotides for toxic C9orf72 RNA

The development of antisense oligonucleotide for toxic C9orf72 RNA is proposed as a potential cure for FTD [1]. Despite the hexanucleotide repeat region being a non-coding sequence, the expanded transcript creates dipeptide protein aggregates by undergoing an independent translation process known as repeat-associated non-ATG-initiated (RAN) translation [8]. Researchers propose developing antisense oligonucleotides that hybridize specifically to the toxic RNA to block RAN translation and prevent further damage [6].

4. Future directions

FTD is a neurodegenerative disorder with no known cure [1]. Various aspects of the C9orf72 gene, which is thought to have an important link to FTD, still remain unknown. As mentioned in section 1.1, the function of the C9ORF72 protein has not yet been identified [1]. Several disease mechanisms have been proposed; however, how exactly the C9orf72 mutant gene leads to FTD has not yet been brought to light. Additionally, the specificity of the commercial antibodies for C9ORF72 protein cannot be guaranteed, and therefore the generation of a more specific antibody is required for more accurate C9OF72 protein measurements [2]. Creation of such antibody will also contribute to the investigation of the C9ORF72 protein localization and possibly the function of C9ORF72 protein itself.
Psychiatric disorders and other neurodegenerative disease such as amyotrophic lateral sclerosis and Huntington’s disease phenocopies have been shown to be the result of C9orf72 repeat expansion [9] [10]. Therefore, finding a cure for FTD by suppressing the effects of the mutant C9orf72 gene could help pave the way for curing many other harmful diseases.

1. van Blitterswijk, M., DeJesus-Hernandez, M., Rademakers, R. (2012). How do C9ORF72 repeat expansions cause amyotrophic lateral sclerosis and frontotemporal dementia: can we learn from other noncoding repeat expansion disorders?. Curr Opin Neurol, 25(6), 689-700
2. DeJesus-Hernandez, M., Mackenzie, I.R., Boeve, B.F., Boxer, A.L., Baker, M., Rutherford, N.J., Nicholson, A.M., Finch, N.A., Flynn, H., Adamson, J., Kouri, N., Wojtas, A., Sengdy, P., Hsiung, G.Y., Karydas, A., Seeley, W.W., Josephs, K.A., Coppola, G., Geschwind, D.H., Wszolek, Z.K., Feldman, H., Knopman, D.S., Petersen, R.C., Miller, B.L., Dickson, D.W., Boylan, K.B., Graff-Radford, N.R., Rademakers, R. (2011). Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron, 72(2), 245-56
3. Waite, A. J., Bäumer, D., East, S., Neal, J., Morris, H. R., Ansorge, O., & Blake, D. J. (2014). Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion. Neurobiology of Aging. doi:10.1016/j.neurobiolaging.2014.01.016
4. Mori K, Weng SM, Arzberger T, May S, Rentzsch K, Kremmer E, Schmid B, Kretzschmar HA, Cruts M, Van Broeckhoven C, Haass C, Edbauer D. (2013). The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science (New York, N.Y.), 339(6125), 1335–1338. doi:10.1126/science.1232927
5. Fratta P, Mizielinska S, Nicoll AJ, Zloh M, Fisher EM, Parkinson G, Isaacs AM. (2012). C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms RNA G-quadruplexes. Sci Rep, 2(1016), 1-6
6. Pliner, H. A., Mann, D. M. & Traynor, B.J. (2014). Searching for Grendel: origin and global spread of the C9ORF72 repeat expansion. Acta Neuropathologica, 127(3), 391-396. doi: 10.1007/s00401-014-1250-x
7. Zamiri, B., Reddy, K., Macgregor, R.B. Jr, Pearson, C.E. (2014). TMPyP4 Porphyrin Distorts RNA G-quadruplex Structures of the Disease-associated r(GGGGCC)n Repeat of the C9orf72 Gene and Blocks Interaction of RNA-binding Proteins. J Biol Chem, 289(8), 4653-9
8. Mizielinska, S., Lashley, T., Norona, F. E., Clayton, E. L., Ridler, C. E., Fratta, P., & Isaacs, A. M. (2013). C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci. Acta Neuropathologica, 126(6), 845–857. doi:10.1007/s00401-013-1200-z
9. Devenney E, Hornberger M, Irish M, Mioshi E, Burrell J, Tan R, Kiernan MC, Hodges JR. (2014). Frontotemporal Dementia Associated With the C9ORF72 Mutation: A Unique Clinical Profile. JAMA Neurology, 71(3), 331–339. doi:10.1001/jamaneurol.2013.6002
10. Hensman Moss, D.J. Poulter, M., Beck J., Hehir J., Polke, J.M., Campbell, T., … Tabrizi, S. J. (2014). C9orf72 expansions are the most common genetic cause of Huntington disease phenocopies. Neurology, 82(4), 292-299. doi: 10.1212/WNL.0000000000000061

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