15 Apr 2022

In Huntington’s disease, the longer the CAG trinucleotide expansion in a person's huntingtin gene, the sooner his or her motor symptoms start. However, the length of inherited repeats alone does not dictate age at onset (AAO). Elongation or contraction of the expansion in somatic cells can speed or slow disease progression, as well. How would that work? In the April 4 Nature Neuroscience, scientists led by Thomas Massey and Lesley Jones at Cardiff University, Wales, U.K., report that certain rare coding mutations in FANCI-associated nuclease 1 blunt the enzyme's activity in cultured cells, leading to rapid CAG repeat expansion. People who carry these variants develop symptoms earlier than expected. On the other hand, other coding variants, in the FAN1 protein-protein interaction domain, correlated with later-than-expected onset. These amino acid changes may alter binding to enzymes that tack nucleotides onto CAG repeats, preventing its expansion. Together, the findings help explain previous genome-wide associations that linked common FAN1 variants with either earlier or later AAO than predicted by inherited expansion alone.

“This is an elegant use of human genetics, taking information from patients down to human cell models, to provide robust evidence for the role of FAN1 nuclease in CAG repeat stability and expansion,” Harry Orr, University of Minnesota, Minneapolis, told Alzforum. Christopher Ross of Johns Hopkins University, Baltimore, agreed. “This beautiful paper provides a striking confirmation that somatic [DNA] instability is important for Huntington’s pathogenesis,” he said. “It shows concrete biochemical evidence that the activity of an enzyme expressed at a genetic locus implicated in HD modifies the age of disease onset,” Ross added.

Previous genome- and transcriptome-wide association studies had correlated common FAN1 variants or expression with shifts in AAO, suggesting that DNA repair enzymes in somatic cells influence disease progression (Gusella et al., 2019; Lee et al., 2022; Goold et al., 2019). In fact, knocking out FAN1 in a mouse model of HD sped up CAG expansion in the cortex and striatum, supporting the hypothesis (Loupe et al., 2020; reviewed by Jones et al., 2017). Elongation of CAG repeats had also been reported in cortical and striatal neurons from HD cases (Shelbourne et al., 2007; Swami et al., 2009). Could FAN1 be to blame?

To find out, co-first authors Branduff McAllister and Jasmine Donaldson assessed the function of FAN1 variants found in people with extremely early or late AAO. To identify as many of these mutants as possible, they obtained exome sequences of 500 REGISTRY-HD and 238 PREDICT-HD participants. The former, a European cohort, enrolled people with HD symptoms; the latter, a U.S.-based study, enrolled healthy HTT mutation carriers and followed them until symptom onset. The scientists picked equal parts participants whose AAO was earlier, or later, than predicted. All were of European descent.  

Just as had prior GWAS, exome sequencing identified FAN1 variants that correlated with AAO. Fourteen rare coding mutations were predicted to cripple enzyme function because their combined annotation-dependent depletion score was above 20. CADD predicts how bad a mutation might be based on how conserved the affected amino acid is in evolution and how likely the mutation is to affect structure and function. Ten variants associated with earlier, four with delayed disease onset. Eight of the early onset mutations lay in FAN1’s DNA-binding or catalytic domains, implying that loss of its nuclease activity speeds HD onset. Intriguingly, all four late-onset FAN1 variants were within the protein-protein interaction domain.

How do these variants have opposing effects? The authors believe this comes down to how FAN1 works. The nuclease has several functions. It cuts off 5' DNA fragments that fail to base-pair if two strands of DNA reanneal out of phase with each other. This function is also how the enzyme repairs inter-strand crosslinks. But in addition, FAN1 liaises with other proteins that can bind and stabilize DNA structures ripe for repeat expansions.

Cultured human cells without FAN1 had repair errors and unstable genomes (Smogorzewska et al., 2010; MacKay et al., 2010; Kratz et al., 2010). The scientists thought the same might be true in people with FAN1 variants (see image below). They predicted that loss of FAN1 function would let DNA expansions grow and disease speed up, while variants in the protein-protein binding domain might allow FAN1 to sequester DNA repair proteins that promote the addition of nucleotides, thereby blocking repeat expansion.

FAN1 and Expansion. In somatic cells, CAG expansions longer than 35 repeats (red) and their complementary sequence (blue) can buckle to form a plus-sign structure to which DNA mismatch repair proteins, such as Msh3, bind (1). The proteins cleave the DNA (2), which partially reanneals to form either long DNA overhangs or gaps (3). FAN1 chops off the flaps to shorten the repeat sequence (4a), while DNA polymerases fill the gaps, lengthening the expansion (4b). [Courtesy of McAllister et al., Nature Neuroscience, 2022.]

To test these ideas, the scientists measured the effect of the mutations on FAN1 activity. They expressed FAN1 variants in bacteria, purified the enzymes, then measured how well they chopped off a flap from a short DNA segment. All six variants tested stunted nuclease activity. Four in the DNA-binding or nuclease sites that were found in people with earlier-than-expected AAO were less than half as active as wild-type FAN1. Two variants in the protein-protein interaction domain that were found in people with delayed AAO, D702E and K794R, also had reduced nuclease activity, but not by as much (see image below). The authors hypothesize that the PPI mutations may block binding of FAN1 to DNA repair enzymes required to add trinucleotide segments, inadvertently preventing elongation of, or even shortening, CAG repeats (see image above).

Could faulty FAN1 hasten CAG repeat expansion in somatic cells? Indeed, when the scientists knocked out FAN1 in induced pluripotent stem cells (iPSCs) from a person with Huntington's, the cells added a CAG unit into the HTT gene 3.5 times faster than did cells with the nuclease. CAG expansion also accelerated in iPSCs carrying the D960A FAN1 mutation, which eliminates nuclease activity but leaves DNA binding intact. Cells with wild-type FAN1 added one repeat in 24 days, while iPSCs with one copy of the mutant did so in 19 days, and cells with two mutant copies, or FAN1 knocked out, 16 days. The same was true when the iPSCs were differentiated into neurons or neural precursor cells. All told, the researchers believe FAN1 nuclease activity puts the brakes on trinucleotide repeat expansion.

The findings may have implications beyond HD. In people with spinocerebellar ataxias, another group of CAG-repeat diseases, FAN1 variants came with altered AAO, as well (Bettencourt et al., 2016). “While the influence of somatic instability on age at onset is still unknown for SCA, implicating FAN1 activity sparks my interest,” Orr said.

There are hints that non-CAG repeats might likewise be modulated. In a mouse model of Fragile X syndrome, knocking out FAN1 doubled the length of CGG repeats in the FMR1 gene in brain tissue (Zhao et al., 2018). 

What about larger repeats, such as hexanucleotide expansions in the C9ORF72 gene that cause frontotemporal dementia and amyotrophic lateral sclerosis? “It’s quite likely that there are conserved mechanisms of repeat expansion throughout the genome,” Massey told Alzforum. Orr and Ross agreed. “If not FAN1, then another nuclease may be involved,” Orr speculated.

Could targeting FAN1 slow down Huntington's? Massey and Ross think the enzyme itself is a bad drug target because it would need to be upregulated. They favor inhibiting proteins that interact with FAN1. “MSH3 is an attractive drug target because knockout mice are viable and do not develop cancer, a worry when tinkering with DNA repair enzymes,” Massey said. Furthermore, in a mouse model of myotonic dystrophy, knocking out MSH3 slowed CTG-repeat expansion in the DMPK gene (Foiry et al., 2006). Triplet Therapeutics in Cambridge, Massachusetts, is developing an antisense oligonucleotide against MSH3, called TTX-3360, to treat myotonic dystrophy, HD, and SCA. According to the company website, they plan to file an investigational new drug application for HD in mid-2022.—Chelsea Weidman Burke

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References

Paper Citations

1. Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium. Electronic address: gusella@helix.mgh.harvard.edu, Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium. CAG Repeat Not Polyglutamine Length Determines Timing of Huntington's Disease Onset. Cell. 2019 Aug 8;178(4):887-900.e14. PubMed.
2. Lee JM, Huang Y, Orth M, Gillis T, Siciliano J, Hong E, Mysore JS, Lucente D, Wheeler VC, Seong IS, McLean ZL, Mills JA, McAllister B, Lobanov SV, Massey TH, Ciosi M, Landwehrmeyer GB, Paulsen JS, Dorsey ER, Shoulson I, Sampaio C, Monckton DG, Kwak S, Holmans P, Jones L, MacDonald ME, Long JD, Gusella JF. Genetic modifiers of Huntington disease differentially influence motor and cognitive domains. Am J Hum Genet. 2022 May 5;109(5):885-899. Epub 2022 Mar 23 PubMed.
3. Goold R, Flower M, Moss DH, Medway C, Wood-Kaczmar A, Andre R, Farshim P, Bates GP, Holmans P, Jones L, Tabrizi SJ. FAN1 modifies Huntington's disease progression by stabilizing the expanded HTT CAG repeat. Hum Mol Genet. 2019 Feb 15;28(4):650-661. PubMed.
4. Loupe JM, Pinto RM, Kim KH, Gillis T, Mysore JS, Andrew MA, Kovalenko M, Murtha R, Seong I, Gusella JF, Kwak S, Howland D, Lee R, Lee JM, Wheeler VC, MacDonald ME. Promotion of somatic CAG repeat expansion by Fan1 knock-out in Huntington's disease knock-in mice is blocked by Mlh1 knock-out. Hum Mol Genet. 2020 Nov 4;29(18):3044-3053. PubMed.
5. Jones L, Houlden H, Tabrizi SJ. DNA repair in the trinucleotide repeat disorders. Lancet Neurol. 2017 Jan;16(1):88-96. PubMed.
6. Shelbourne PF, Keller-McGandy C, Bi WL, Yoon SR, Dubeau L, Veitch NJ, Vonsattel JP, Wexler NS, , Arnheim N, Augood SJ. Triplet repeat mutation length gains correlate with cell-type specific vulnerability in Huntington disease brain. Hum Mol Genet. 2007 May 15;16(10):1133-42. PubMed.
7. Swami M, Hendricks AE, Gillis T, Massood T, Mysore J, Myers RH, Wheeler VC. Somatic expansion of the Huntington's disease CAG repeat in the brain is associated with an earlier age of disease onset. Hum Mol Genet. 2009 Aug 15;18(16):3039-47. Epub 2009 May 23 PubMed.
8. Smogorzewska A, Desetty R, Saito TT, Schlabach M, Lach FP, Sowa ME, Clark AB, Kunkel TA, Harper JW, Colaiácovo MP, Elledge SJ. A genetic screen identifies FAN1, a Fanconi anemia-associated nuclease necessary for DNA interstrand crosslink repair. Mol Cell. 2010 Jul 9;39(1):36-47. PubMed.
9. MacKay C, Déclais AC, Lundin C, Agostinho A, Deans AJ, MacArtney TJ, Hofmann K, Gartner A, West SC, Helleday T, Lilley DM, Rouse J. Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2. Cell. 2010 Jul 9;142(1):65-76. PubMed.
10. Kratz K, Schöpf B, Kaden S, Sendoel A, Eberhard R, Lademann C, Cannavó E, Sartori AA, Hengartner MO, Jiricny J. Deficiency of FANCD2-associated nuclease KIAA1018/FAN1 sensitizes cells to interstrand crosslinking agents. Cell. 2010 Jul 9;142(1):77-88. PubMed.
11. Bettencourt C, Hensman-Moss D, Flower M, Wiethoff S, Brice A, Goizet C, Stevanin G, Koutsis G, Karadima G, Panas M, Yescas-Gómez P, García-Velázquez LE, Alonso-Vilatela ME, Lima M, Raposo M, Traynor B, Sweeney M, Wood N, Giunti P, SPATAX Network, Durr A, Holmans P, Houlden H, Tabrizi SJ, Jones L. DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases. Ann Neurol. 2016 Jun;79(6):983-90. Epub 2016 May 6 PubMed.
12. Zhao XN, Usdin K. FAN1 protects against repeat expansions in a Fragile X mouse model. DNA Repair (Amst). 2018 Sep;69:1-5. Epub 2018 Jul 5 PubMed.
13. Foiry L, Dong L, Savouret C, Hubert L, te Riele H, Junien C, Gourdon G. Msh3 is a limiting factor in the formation of intergenerational CTG expansions in DM1 transgenic mice. Hum Genet. 2006 Jun;119(5):520-6. Epub 2006 Mar 22 PubMed.

External Citations

1. TTX-3360

Further Reading

Papers

1. Kim KH, Hong EP, Shin JW, Chao MJ, Loupe J, Gillis T, Mysore JS, Holmans P, Jones L, Orth M, Monckton DG, Long JD, Kwak S, Lee R, Gusella JF, MacDonald ME, Lee JM. Genetic and Functional Analyses Point to FAN1 as the Source of Multiple Huntington Disease Modifier Effects. Am J Hum Genet. 2020 Jul 2;107(1):96-110. Epub 2020 Jun 25 PubMed.
2. Moss DJ, Pardiñas AF, Langbehn D, Lo K, Leavitt BR, Roos R, Durr A, Mead S, TRACK-HD investigators, REGISTRY investigators, Holmans P, Jones L, Tabrizi SJ. Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study. Lancet Neurol. 2017 Sep;16(9):701-711. Epub 2017 Jun 20 PubMed.
3. Flower M, Lomeikaite V, Ciosi M, Cumming S, Morales F, Lo K, Hensman Moss D, Jones L, Holmans P, TRACK-HD Investigators, OPTIMISTIC Consortium, Monckton DG, Tabrizi SJ. MSH3 modifies somatic instability and disease severity in Huntington's and myotonic dystrophy type 1. Brain. 2019 Jun 19; PubMed.