loss-of-function mutations in the c9orf72 mouse ortholog ... · of both komp and neo-deleted...

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Loss-of-function mutations in the C9ORF72 mouseortholog cause fatal autoimmune diseaseAaron Burberry,1,2 Naoki Suzuki,1,2 Jin-Yuan Wang,1,2 Rob Moccia,1,2 Daniel A. Mordes,1,2,3

Morag H. Stewart,1,4 Satomi Suzuki-Uematsu,1,2 Sulagna Ghosh,1,2 Ajay Singh,1,2

Florian T. Merkle,1,2 Kathryn Koszka,1,2 Quan-Zhen Li,5 Leonard Zon,1,6 Derrick J. Rossi,1,4,6

Jennifer J. Trowbridge,7 Luigi D. Notarangelo,6,8 Kevin Eggan1,2*

C9ORF72 mutations are found in a significant fraction of patients suffering from amyotrophic lateral sclerosisand frontotemporal dementia, yet the function of the C9ORF72 gene product remains poorly understood. Weshow that mice harboring loss-of-function mutations in the ortholog of C9ORF72 develop splenomegaly, neu-trophilia, thrombocytopenia, increased expression of inflammatory cytokines, and severe autoimmunity, ultimatelyleading to a high mortality rate. Transplantation of mutant mouse bone marrow into wild-type recipients was suf-ficient to recapitulate the phenotypes observed in the mutant animals, including autoimmunity and prematuremortality. Reciprocally, transplantation of wild-type mouse bone marrow into mutant mice improved their pheno-type. We conclude that C9ORF72 serves an important function within the hematopoietic system to restrict inflam-mation and the development of autoimmunity.

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INTRODUCTION
Amyotrophic lateral sclerosis (ALS) is characterized by the progressivedegeneration of motor neurons, resulting in paralysis and death (1).Genetic findings suggest that ALS can result from mutations in genesacting in several cellular processes (2). To date, the most commonmutation found in ALS patients, as well as in frontotemporal demen-tia (FTD), is a hexanucleotide repeat expansion in the first intron ofC9ORF72 (3). This mutation is present in 4 to 8% of patients with spo-radic ALS (4). The repeat expansion is also found in 40% of familial ALScases from Europe and the United States and is even more prevalentin Scandinavia (3, 4). Identification of unaffected elderly carriers sug-gests that the mutation’s effects are incompletely penetrant (5).

The expanded GGGGCC repeat in C9ORF72 has been proposedto mediate its effects through one mechanism or a combination ofthree mechanisms: the creation of long repetitive RNAs (3, 6), thetranslation of these RNAs into toxic repetitive dipeptides (7), or thesilencing of the mutant allele (3, 8, 9). Histological and gene expres-sion studies indicate that all three effects of the repeat expansion man-ifest themselves in individuals harboring it. However, determiningwhich effect or effects of the mutation contribute to the degenerativephenotypes seen in patients remains unresolved. The normal proteinproduct of C9ORF72 contains a DENN domain (10) and may act asa guanine nucleotide exchange factor (11). Previous studies in fishand worms suggested that C9ORF72might function in the nervous sys-tem (8, 12). We found that although the murine ortholog of C9ORF72(3110043O21Rik, hereafter referred to as C9orf72) is widely expressed, itwas enriched in motor neurons known to degenerate in ALS (13). How-

1Harvard Stem Cell Institute, Department of Stem Cell and Regenerative Biology, HarvardUniversity, Cambridge, MA 02138, USA. 2Stanley Center for Psychiatric Research, BroadInstitute of MIT and Harvard, Cambridge, MA 02142, USA. 3Department of Pathology,Massachusetts General Hospital, Boston, MA 02114, USA. 4Program in Cellular and Molec-ular Medicine, Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA02115, USA. 5Departments of Immunology and Internal Medicine, University of TexasSouthwestern Medical Center, Dallas, TX 75390, USA. 6Harvard Medical School, Boston,MA 02115, USA. 7The Jackson Laboratory for Mammalian Genetics, Bar Harbor, ME04609, USA. 8Division of Immunology, Boston Children’s Hospital, Boston, MA 02115, USA.*Corresponding author. Email: [email protected]

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ever, a recent study demonstrated that conditional excision of C9orf72in cells of the nervous system had no overt effects, raising the questionof whether it plays any role in the nervous system or overall health (14).

Resolving the function of the endogenous C9ORF72 gene productremains important because it could provide insight into whether thereduction in gene product found in patients with the repeat expansioncontributes to their disease (3, 8, 9). Furthermore, efforts are proceed-ing to develop therapeutic approaches for knocking down the gain-of-function products of the repeat expansion (9). Because knockdownstrategies could inadvertently depress transcription of the normal geneproduct, it will be critical to understand the phenotypic ramificationsof long-term depression of gene expression from this locus.

Here, we report an initial study of mice harboring loss-of-functionmutations in C9orf72. We found that homozygous mutant animalsproduced through homologous recombination, as well as CRISPR (clus-tered regularly interspaced short palindromic repeats)/Cas9 targeting,developed several classical features of autoimmunity. Transplantationstudies demonstrated that mutant bone marrow cells were sufficientto cause autoimmunity and premature mortality when placed into other-wise wild-type recipients. Thus, we conclude that the gene productencoded by C9orf72 likely acts within the hematopoietic system toplay an important role in the promotion of immunological tolerance.

RESULTS

C9orf72 loss-of-function mutations cause premature mortalityWe recently identified the mouse ortholog of C9ORF72 and generatedheterozygous mice harboring a LacZ insertion replacing exons 2 to 6of the gene on an inbred C57BL/6 background (13) (Fig. 1A). Becausethis targeting event eliminated highly conserved 5′ exons of C9orf72,we reasoned that it might result in a loss of gene function, allowing theimportance of this gene to be investigated. After intercrossing hetero-zygous (+/−) animals, we found that mice of the three expected geno-types were recovered at expected Mendelian frequencies (Fig. 1B). Wedesignated this strain “KOMP” because the gene targeting to create

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this allele was originally performed by the Knock Out Mouse Project(KOMP) Consortium.

We next sought to use animals from the +/− intercrosses to deter-mine whether the targeted mutation reduced expression of C9orf72.Using exon-spanning primers, we detected a significant, dose-dependentreduction in the abundance of transcript sequences from exons 6through 8 in whole blood of +/− and −/− animals relative to that foundin wild-type controls (Fig. 1C; **P < 0.01). To assess whether the mu-

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tation also resulted in decreased proteinlevels, we used two anti-C9orf72 antibodies,one raised against C-terminal regions of theprotein and the other against its N termi-nus. In each case, a band with the predictedmass of C9orf72 was significantly depletedin mutant animals (Fig. 1, D and E; *P <0.05, **P < 0.01).

There are historical examples of thetranscriptional units encoding drug re-sistance used in gene targeting leadingto off-target phenotypes (15). We there-fore removed the selection cassette usedfor gene targeting by outcrossing C9orf72-targeted KOMP mice with outbred ani-mals that expressed the Cre recombinaseunder the control of the Sox2 promoter(16) (hereafter referred to as “Neo-deleted”)(Fig. 1A and fig. S1A). Intercrosses of +/−Neo-deleted animals resulted in recoveryof +/+, +/−, and −/− animals at the ex-pected Mendelian frequencies (Fig. 1F).Neo-deleted +/− and −/− mice exhibiteda significant and dose-dependent reductionin C9orf72 transcript abundance (Fig. 1G;**P < 0.01). Because proper interpretationof mouse models relies on the fidelity ofthe genetic modifications introduced, weperformed whole-genome sequencing ofrepresentative +/+, +/−, and −/− KOMPanimals, as well as −/− Neo-deleted mice.These studies revealed proper gene target-ing in each strain and the absence of off-target insertions (fig. S1B). We were alsoable to confirm the C57BL/6 inbred natureof KOMP animals and outbred nature ofNeo-deleted animals (fig. S1, C to F).

To determine the effects of C9orf72 lossof function, we intercrossed +/−KOMP ani-mals and monitored littermates for 400 days(Fig. 2A; n = 163; +/+, n = 50; +/−, n =84; −/−, n = 29). At 70 days of age, wenoted no obvious phenotypes (Fig. 2A).However, as the cohort continued to age,the risk of death in both −/− and +/− ani-mals grew to a high level of significance(Fig. 2A; *P < 0.05). We also examinedNeo-deleted mice generated by +/− inter-crosses (Fig. 2B; n = 50; +/+, n = 15; +/−,n = 21; −/−, n = 14). At later ages, we sim-

ilarly observed significantly increased mortality in +/− and −/− mice(Fig. 2B; *P < 0.05). Because C9orf72 has been genetically implicatedin ALS, we asked whether increased mortality in mutant animals wasassociated with neural degeneration. However, we did not find obvi-ous changes in the number of spinal motor neurons (fig. S2). Nor didwe observe differences in the gross histology of the mutant motor cor-tex (fig. S3) or other brain regions of mutant animals (fig. S4). We did,however, observe evidence for increased neural inflammation in the

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Fig. 1. A loss-of-function mutation in the C9orf72 ortholog. (A) The schematic illustrates replacementof exons 2 to 6 to generate the KOMP allele and crosses with Sox2-cre mice to generate the Neo-deleted
allele. 3′ HA, 3′ homology arm; FRT, flippase recognition target. (B) Frequency of genotypes born fromKOMP +/− crosses. (C) C9orf72 expression in KOMP whole blood by quantitative reverse transcription poly-merase chain reaction (RT-PCR). **P < 0.01, Tukey multiple comparisons. (D) Western blotting of corticaltissue from the three KOMP genotypes using anti-C9ORF72 antibodies. (E) Quantification of Western blotbands. *P < 0.05, **P < 0.01, Tukey multiple comparisons. ns, not significant. Ab, antibody. (F) Frequency ofgenotypes born from Neo-deleted +/− crosses. (G) C9orf72 expression in Neo-deleted whole blood byquantitative RT-PCR. **P < 0.01, Tukey multiple comparisons.
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cortex of mutant animals as evidenced by focal staining with anti–GFAP (glial fibrillary acidic protein) antibodies (fig. S5).

We next sought to establish the onset of decline in these mutantanimals, to quantify the duration of their decline, and to better under-stand the causes of their premature death. We weighed mice weeklyand performed necropsy on animals found dead or that we wereobliged to euthanize because of preestablished animal welfare criteria.We noted that although animals of all genotypes initially exhibited


similar weights, as they aged, the weightof both KOMP and Neo-deleted −/− ani-mals plateaued and became significantlylower than that of wild-type controls(Fig. 2, C and D; *P < 0.05). By definingphenotypic onset for each animal as theage at which it reached its maximumbody weight, we were then able to quan-tify the number of days that a givenanimal was burdened by decline beforeits death. Heterozygous animals were bur-dened by decline for, on average, 118 ±67 days (KOMP; n = 13) and 56 ± 38 days(Neo-deleted; n = 10), whereas −/− animalswere burdened by decline for, on average,76 ± 51 days (KOMP; n = 21) and 37 ±29 days (Neo-deleted; n = 9) (Fig. 2E). Dur-ing these studies, we determined that thecauses of demise included cachexia, respira-tory failure, hepatomegaly, dermatitis, inter-nal hemorrhage, lymphocytic stromal cellhyperplasia, severe ataxia, and prolapse(Fig. 2E and fig. S6).

Upon necropsy of end-stage animals,we observed enlarged spleens in KOMP−/− (n = 10 of 11) and Neo-deleted −/−(n = 6 of 6) mice (Fig. 3, A and B). Spleno-megaly can result from any one of severalunderlying pathologies, including infection,myeloproliferative disease, chronic lympho-cytic leukemia, lymphoma, and auto-immunity. To investigate whether any ofthese was the cause of splenomegaly inmutant animals, we visually inspectedday 300 spleens from Neo-deleted animals(+/+, n = 6; +/−, n = 3; −/−, n = 7) andthen subjected them to histological analy-sis. We observed a clear demarcation be-tween red and white pulp regions in +/+and +/− spleens, whereas −/− spleens dis-played a disruption of red and white pulpboundaries (Fig. 3B). We next asked whensplenomegaly first appeared in these ani-mals. We found that day 25 Neo-deleted−/− animals exhibited normal spleen sizes,whereas day 50 −/− animals had developedsignificantly enlarged spleens relative totheir littermates (Fig. 3D; **P < 0.01). Thissignificant increase in spleen weight wasmaintained in day 100, day 200, and day

300 −/− animals (Fig. 3D; **P < 0.01). We reasoned that splenic veinthrombosis and spontaneous microbial infection were less likely causesof splenomegaly in −/− animals because of the synchronicity and pen-etrance at which this phenotype developed.

We next considered the possibility that splenomegaly was the re-sult of lymphoma. In lymphoma, individual clonal populations ofeither B or T cells can dominate the splenic compartment. How-ever, mutant animals displayed an increase in splenocytes derived

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Fig. 2. C9orf72 mutations lead to premature death of mice. Mice harboring loss-of-function muta-tions in C9orf72, generated by homologous recombination using a targeting vector in embryonic stem
cells on a C57BL/6 background (KOMP) and those outcrossed with Sox2-cre–expressing mice to re-move the neomycin cassette (Neo-deleted), were aged for survival studies. (A and B) Kaplan-Meiersurvival curves for (A) KOMP and (B) Neo-deleted mice. *P < 0.05, **P < 0.01, generalized Wilcoxontest. (C and D) Body weight of female (C) KOMP mice and (D) Neo-deleted animals over time. *P <0.05, Dunnett’s multiple comparisons. (E) Cause of death or obligatory euthanasia in KOMP and Neo-deleted mice. “Days of decline” indicates the time between maximum body weight (onset) and deathor obligatory euthanasia.
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from several lineages, including CD19+ B220+ B cells, CD3+ CD4+

T cells, and CD11b+ Ly6G+ neutrophils (Fig. 3, E and F, and fig. S7; *P <0.05, **P < 0.01). Furthermore, analysis of V(D)J recombination inspleens of mutant animals (17) revealed polyclonal populations of B


and T cells, arguing against the type of clo-nal expansion observed in lymphoma (Fig.3G). We also did not observe histologicalchanges in the cellularity of −/− bone mar-row (fig. S8), which can be a site of lym-phocytic transformation (18).

In addition to the nearly ubiquitoussplenomegaly observed in −/− animals, wealso noted that a subset of end-stageKOMP −/− (n = 4 of 21) and Neo-deleted−/− (n = 3 of 9) animals developed grosslyenlarged cervical lymph nodes (fig. S9).Enlarged lymph nodes can result frominflammation, lymphoma, autoimmunity,or an inability of lymphocytes to escape thiscompartment (19, 20). Although we notedmodest changes in the relative proportionof B and T cells, both of these populationswere greatly expanded within enlargedcervical lymph nodes of mutant animals(fig. S9), again arguing against clonaltransformation as an explanation. Instead,we observed an increase in T cell activa-tion state, with a significant reduction inCD62Lhi CD44lo cells and a significant in-crease in CD62Lhi CD44hi and CD62Llo

CD44hi cells when compared to controls(fig. S9; *P < 0.05, **P < 0.01).

Finally, we noted that a small butsignificant subset of both KOMP andNeo-deleted mutant animals developedhepatomegaly (Fig. 2E and fig. S10). His-tological analysis suggested that this liverovergrowth was likely the result of im-mune cell infiltration (fig. S10). Such im-mune infiltrates in liver can be seen in thecontext of both leukemia and autoimmu-nity (21). In this case, infiltrating hemato-poietic cells appeared morphologicallydiverse, rather than clonal in nature, whichagain seemed less parsimonious with thediagnosis of leukemia and more consistentwith chronic inflammation (fig. S10).

Changes in peripheral bloodwere consistent with autoimmunitynot leukemiaAssessing the composition of peripheralblood can yield insight into pathways thatdisrupt the hematopoietic system.We there-fore performed whole blood cell countson mutant mice greater than 300 daysof age (Fig. 4, A to H). Numbers of whiteblood cells, platelets, and RBCs in

KOMP +/+ and Neo-deleted +/+ animals were similar to historicranges in inbred mouse strains (22). In contrast, total white blood cellcounts were modestly but significantly elevated in KOMP −/− and Neo-deleted −/− mice (Fig. 4A; **P < 0.01), which was due to a significant

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Fig. 3. Identity of cells within the enlarged mutant spleens of Neo-deleted mice. (A) Spleens fromday 300 Neo-deleted animals. Scale bar, 1 cm. (B) Hematoxylin and eosin staining of spleens from
Neo-deleted mice at day 300. Scale bar, 500 mm (i) and 50 mm (ii). (C and D) Quantification of spleenweight in (C) end-stage KOMP and Neo-deleted animals and (D) aged Neo-deleted animals. (E) Splenocytecounts from day 200 Neo-deleted mice. (F) Quantification of splenocyte subsets in day 200 Neo-deletedmice. NKs, natural killer cells. DCs, dendritic cells. (C, E, and F) *P < 0.05, **P < 0.01, Tukey multiple com-parisons. (D) *P < 0.05, **P < 0.01, Student’s t test. ns, not significant. (G) PCR analysis of T and B cellclonality in the spleens of day 400 Neo-deleted mice. LN2 and LN3 represent embryonic stem cells gen-erated by nuclear transfer from lymph node–derived T cells that harbor monoclonal TCRb (T cell receptorb) rearrangements.
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increase in the number of circulating neutrophils (Fig. 4B; **P < 0.01).We observed no significant changes in the relative proportion ofperipheral lymphocytes (Fig. 5C) or monocytes in mice of differinggenotypes (Fig. 4D).

We also noted that platelet count was significantly reduced inKOMP −/− and Neo-deleted −/− animals (Fig. 4E; **P < 0.01) and thatthere was a modest but significant reduction of RBCs in KOMP −/− andNeo-deleted −/− (Fig. 4F; **P < 0.01) animals that was accompanied by amodest but significant reduction in hematocrit (Fig. 4G; **P < 0.01).Mean corpuscular volume was also reduced in KOMP −/− and Neo-deleted −/− animals (Fig. 4H; **P < 0.01), consistent with modest micro-cytic anemia. Although there can be many causes for changes in red celland platelet counts, similar phenotypes are seen in autoimmune hemo-lytic anemia and thrombocytopenic purpura, in which RBCs or platelets,respectively, are targeted by autoantibodies, leading to their destruction (23).

To understand how changes in the peripheral blood developed, webled Neo-deleted animals (n = 163) ranging from 50 to 200 days ofage (Fig. 4, I to K; +/+, n = 49; +/−, n = 65; −/−, n = 49). We notedthat platelet count was already reduced by day 50 in −/−mice and was


persistently depressed in older animals(Fig. 4K; *P < 0.05). Although young ani-mals displayed normal neutrophil andwhite cell numbers, at 100 days of age,−/− mice began to exhibit a persistentand significant increase in neutrophil num-bers (Fig. 4J; **P < 0.01), which was theprimary contributor to a significant in-crease in total white cell counts (Fig. 4I;*P < 0.05). In contrast, numbers of lym-phocytes in the blood remained unchangedrelative to wild-type controls. Thus, neutro-philia, thrombocytopenia, and splenomegalywere highly penetrant phenotypes, whichall developed between days 50 and 100 in−/− animals, suggesting an interconnectedpathological relationship. In contrast, en-larged lymph nodes and immune cell infil-trates in the liver were observed later and atlower frequencies in mutant animals, sug-gesting that they could be secondary ef-fects of changes to the hematopoieticcompartment.

Loss of C9orf72 causes early andchronic cytokine inductionIf the splenic and blood phenotypes thatwe observed in −/− animals developed inresponse to a chronic inflammatory pro-cess, we reasoned that their emergencemight be associated with an increasedabundance of inflammatory chemokinesand cytokines. To test this idea, we mea-sured the levels of 36 chemokines and cy-tokines in plasma from day 300 KOMP(Fig. 5A; +/+, n = 18; +/−, n = 8; −/−,n = 11) and Neo-deleted animals (Fig. 5B;+/+, n = 3; +/−, n = 11; −/−, n = 4). Eigh-teen of 36 cytokines and chemokines were

significantly elevated in both KOMP −/− and Neo-deleted −/− animalsrelative to +/+ controls, including IL-22 (interleukin-22), IL-28, IL-23,IL-6, MCP-1 (monocyte chemoattractant protein 1), IL-31, IL-5, IL-10,IL-1b, IL-15/IL-15R (IL-15 receptor), IFN-g (interferon-g), IL-3, GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-17A,IFN-a, MIP-1B (macrophage inflammatory protein-1B), LIF (leukemiainhibitory factor), and GROa (growth-related oncogene a) (Fig. 5, Aand B; *P < 0.05, **P < 0.01). No cytokines or chemokines were signif-icantly changed in KOMP +/− mice relative to controls (Fig. 5A), al-though we did note that IL-28 was significantly elevated and IL-2 wassignificantly reduced in +/− Neo-deleted animals relative to controls(Fig. 5B; *P < 0.05). We next analyzed cytokine levels in cohorts of miceat earlier time points. We found that already at 50 days of age, IL-31,IL-15/IL-15R, and GM-CSF were significantly elevated in Neo-deleted−/−mice relative to wild-type mice (fig. S11; *P < 0.05). By 100 days ofage, Neo-deleted −/− animals displayed significantly increased levels of19 of 36 cytokines and chemokines tested, fully recapitulating the pat-tern observed in older animals at both days 200 and 300 (fig. S11; *P <0.05, **P < 0.01). Thus, the development of an inflammatory cytokine

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Fig. 4. Mice with C9orf72 mutations develop hematological phenotypes. (A to H) Peripheral bloodcounts assessed for day 300+ KOMP and Neo-deleted animals. *P < 0.05, **P < 0.01, Tukey mul-
tiple comparisons. ns, not significant. (A) White blood cells. (B) Neutrophils. (C) Lymphocytes. (D)Monocytes. (E) Platelets. (F) Red blood cells (RBCs). (G) Hematocrit. (H) Mean corpuscular volume.(I to K) Peripheral blood counts assessed for aged Neo-deleted animals. (I) White blood cells. (J) Neu-trophils. (K) Platelets.
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profile was an early event in mutant animals that either preceded orwas coincident with other blood phenotypes and a decline in health.

Mutant animals rapidly develop persistent autoimmunityWe next considered the possibility that the changes in cytokine levelsthat we found were due to autoimmunity. Autoantibodies targetingdouble-stranded DNA (dsDNA) are a common diagnostic markerof autoimmune syndromes, including systemic lupus erythematosus


(24). We found significant accumulationof anti-dsDNA antibody activity between50 and 100 days of age in Neo-deleted−/− animals (Fig. 5D; **P < 0.01), whichwas sustained in plasma from KOMP−/− and Neo-deleted −/− animals greaterthan 300 days of age (Fig. 5C; *P < 0.05,**P < 0.01).

Although the accumulation of anti-dsDNA antibodies occurs under con-ditions of autoimmunity, it can also beindicative of rapid but transient cell death(25). To distinguish between these twopossibilities, we used established auto-antigen microarrays to test the plasmaof day 300 Neo-deleted littermates forthe presence and abundance of immuno-globulin M (IgM) and IgG antibodies tar-geting 124 autoantigens from disparatecell types (26) (Fig. 5, E and F). In −/−plasma, IgM autoantibodies targeting117 of 124 antigens were significantly ele-vated relative to controls. Similarly, IgGautoantibodies targeting 113 of 124 anti-gens were significantly elevated in −/−animals (table S1; *P < 0.05). In +/− plas-ma, IgM autoantibodies targeting 1 of124 antigens [liver kidney microsomaltype 1 (LKM1)] were significantly ele-vated and IgG autoantibodies targeting2 of 124 antigens (Collagen II and Mi-2)were significantly elevated relative to con-trols (table S1; *P < 0.05). Hierarchicalclustering revealed that the pattern ofIgM autoantibody activity in five of six−/− animals was significantly distinct from+/+ controls and two of four +/− animals(Fig. 5E). Intriguingly, the other two of four+/− animals displayed an intermediate pat-tern of IgM autoantibody reactivity, clus-tering with the remaining −/− mice (Fig.5E). Clustering based on IgG-recognizedautoantigens placed all mutants in one dis-tinct group, with no obvious distinction be-tween +/− and +/+ found in this case (Fig.5F). Thus, we conclude that loss of C9orf72results in the accumulation of a wide arrayof self-reactive antibodies, which is indica-tive of an autoimmune phenotype.

Because T regulatory cells restrict in-
flammation, we measured the percentage of CD25+ CD4+ cells, whichare enriched for this T cell subtype, in spleens from 400-day-old Neo-deleted animals and found that CD25+ CD4+ cells were significantlyelevated in −/− spleens relative to +/+ animals (fig. S12; **P < 0.01).
C9orf72 promotes tolerance in the immune systemThe ImmGen database (27) indicates that C9ORF72 and its murineortholog are expressed in blood cells. We therefore asked whether

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Fig. 5. Mice with C9orf72 mutations show increased cytokines, chemokines, and autoantibodies.(A and B) Analysis of 36 plasma cytokines and chemokines in day 300+ (A) KOMP and (B) Neo-deleted
animals. *P < 0.05, **P < 0.01, Tukey multiple comparisons. (C and D) Anti-dsDNA antibody reactivity inplasma of (C) day 300+ KOMP and Neo-deleted animals and (D) aged Neo-deleted animals. *P < 0.05,**P < 0.01, Tukey multiple comparisons. ns, not significant. (E and F) Plasma from day 300+ Neo-deletedmice assessed for (E) IgM and (F) IgG reactivity against 124 self-antigens (26). Unsupervised hierarchicalclustering grouped individual animals (x axis) and self-antigens (y axis).
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C9orf72 acts within the hematopoieticcompartment. To address this, we per-formed bone marrow transplantation(BMT) between animals of distinct geno-types. Because of their inbred nature, weperformed reciprocal BMT experimentsusing KOMP mice. To serve as pheno-typic negative and positive controls, wetransplanted −/− bone marrow into −/−recipients (−/−→−/−) (n = 6) and +/+marrow into +/+ recipients (WT→WT)(Fig. 6A). To test whether a −/− geno-type in bone marrow–derived cells wassufficient to cause autoimmunity, we trans-planted −/− bone marrow into +/+ recipi-ents (−/−→WT) (n = 12). Reciprocally,we asked whether loss of C9orf72 in theblood was necessary for the develop-ment of autoimmunity by attemptingto ameliorate the phenotype of −/− ani-mals with transplantation of +/+ marrow(WT→−/−) (n = 7). B6.SJL-PtprcaPepcb/BoyJ mice were used as wild-type bonemarrow donors and recipients to facili-tate tracking of reconstitution efficiency(Fig. 6A). No animals perished imme-diately after BMT. By 17 weeks, 29 of30 transplanted animals exhibited >90%reconstitution of CD45+ cells, includingCD11b+ Ly6G− monocytes, CD11b+ Ly6G+

neutrophils, and B220+ B cells (Fig. 6, Band C, and fig. S13), indicating that en-graftment of donor hematopoietic cellswas efficient and sustained. However, wedid note that CD3+ T cell reconstitutionwas incomplete, ranging from 40 to 80%donor-derived cells (Fig. 6, B and C, andfig. S13), which can be observed becauseof the relative radio resistance and long-lived nature of this cell type (28).

All WT→WT animals survived be-yond 460 days of age (Fig. 6D) and pro-gressively gained weight (Fig. 6E). In linewith our previous findings (Fig. 2), we not-ed that mice lacking C9orf72 (−/−→−/−)failed to gain weight as fast as their wild-type (WT→WT) counterparts, a differ-ence that became significant by 174 daysof age (Fig. 7E; *P < 0.05). Moreover, all−/−→−/− animals (n = 6 of 6) died by350 days, with a median survival of293 days (Fig. 6D; *P < 0.05), and thecauses of death or obligatory euthanasiain −/−→−/− animals were consistentwith those observed previously (Fig. 2E,fig. S13, and video 1). At end stage,−/−→−/− mice had developed spleno-megaly (Fig. 6F; **P < 0.01), neutrophilia

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Fig. 6. C9orf72 acts in bone marrow–derived cells to prevent autoimmunity. (A) Wild-type (WT) orC9orf72-deficient animals were lethally irradiated at day 110 and reconstituted with WT or mutant
bone marrow. Recipient mice were regularly weighed, monitored for survival, and bled for whole-bloodcell counts and plasma analyses, and animals were necropsied at end stage. (B and C) Quantification offlow cytometry–based assessment of 17-week posttransplant peripheral blood reconstitution. (B, C, andF to K) Each dot represents one mouse. (D) Survival curves for transplanted mice. *P < 0.05, generalizedWilcoxon test. IR, irradiation. (E) Average body weight ± SEM. *P < 0.05, Dunnett’s multiple compar-isons. (F) End-stage spleen weight. (G) End-stage peripheral blood neutrophil counts. (H) End-stageperipheral blood platelet counts. (I) End-stage hematocrit. (J and K) Plasma anti-dsDNA antibody ac-tivity in animals at (J) day 230 (D230) and (K) end stage. (F to K) *P < 0.05, **P < 0.01, Tukey multiplecomparisons. ns, not significant.
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(Fig. 6G; **P < 0.01), thrombocytopenia(Fig. 6H; **P < 0.01), and anemia (Fig. 6I;**P < 0.01). Anti-dsDNA antibody activ-ity was significantly elevated in −/−→−/−at an age that preceded mortality (day230) (Fig. 6J; **P < 0.01), as well as atend stage (Fig. 6K; **P < 0.01). Thus, le-thal irradiation and BMT did not affectthe development of autoimmunity in−/− animals.

We asked which of the phenotypesfound in −/− animals could be observedin +/+ animals receiving −/− bone marrow.We found that −/−→WT animals weresignificantly smaller than WT→WT ani-mals at 206 days of age, after which theirbody weight plateaued (Fig. 6E; *P < 0.05).Strikingly, 9 of 12 −/−→WT mice died by460 days of age with a median survival of443 days (Fig. 6D; *P < 0.05 relative toWT→WT; video 2). At end stage, all−/−→WT (n = 9 of 9) had larger spleens(Fig. 6F; **P < 0.01), whereas a subset of−/−→WT mice (3 of 12) had developedenlarged cervical lymph nodes (fig. S13).Relative to WT→WT mice, −/−→WTanimals had similar numbers of neutro-phils (Fig. 6G) but displayed a signifi-cantly lower platelet count (Fig. 6H; **P <0.01) and had significantly reduced he-matocrit (Fig. 6I; **P < 0.01). IL-22, IL-31,and MIP-1B were significantly ele-vated in −/−→WT animals relative toWT→WT at end stage (fig. S13; *P <0.05). Anti-dsDNA antibody activity wassimilar in −/−→WT and WT→WT ani-mals at day 230 (Fig. 6J), yet by end stage,−/−→WT displayed significantly elevatedanti-dsDNA antibody activity (Fig. 6K;*P < 0.05).

Next, we asked whether transplanta-tion of WT bone marrow into −/− ani-mals could improve their phenotype. By230 days of age, WT→−/− mice hadgained more weight than their −/−→−/−counterparts (Fig. 6E; *P < 0.05). Moreover,WT→−/− animals lived significantly longerthan −/−→−/− animals, with the longestlived WT→−/− animals surviving to452 days of age and with a median sur-vival of 340 days, representing a 43-dayextension of life span (Fig. 6D; *P < 0.05relative to −/−→−/−; video 3). Splenomegalywas apparent in all WT→−/− animals atend stage, but on average, their spleenswere smaller than −/−→−/− mice (Fig.6F; **P < 0.01). Relative to −/−→−/− ani-mals, WT→−/− mice had significantly

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in several distinct mutations. (B) DNA sequences from CRISPR/Cas9-targeted mice indicated that 23 of24 animals harbored a stop codon in exon 4 of the C9orf72 gene. (C) Survival of CRISPR/Cas9-targetedmice. (D) Spleens from a CRISPR/Cas9-targeted mouse at end stage and an age-matched C57BL/6control. (E) Spleen weights for CRISPR/Cas9 mutant and KOMP mice. (F) Hematocrit for day 300+CRISPR/Cas9-targeted animals. (G) Plasma anti-dsDNA antibody activity in day 300+ CRISPR/Cas9-targetedanimals. (E to G) Each dot represents one mouse. *P < 0.05, **P < 0.05, Dunnett’s multiple comparisons. ns,not significant. (H) Mice harboring a 38-nucleotide deletion in exon 4 of C9orf72 were bred to heterozy-gosity (+/del) and homozygosity (del/del), as visualized by PCR of tail DNA using primers flanking thedeletion site. (I) Plasma anti-dsDNA antibody reactivity in day 150 +/+, +/del, and del/del animals.**P < 0.01, Tukey multiple comparisons. (J) Proposed model for how C9orf72 may act in bone marrow–derived cells to limit fatal immune deregulation.
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fewer neutrophils (Fig. 6G; **P < 0.01) and significantly elevated plate-let counts (Fig. 6H; **P < 0.01), but hematocrit was not significantlyimproved (Fig. 6I). IL-31, IL-6, MIP-1B, IL-10, IL-17A, and IL-15/IL-15R were significantly reduced in WT→−/− relative to −/−→−/− atend stage (fig. S13; *P < 0.05). Anti-dsDNA activity was significantlyreduced in WT→−/− relative to −/−→−/− animals at day 230 (Fig. 6J;**P < 0.01) and at end stage (Fig. 6K; *P < 0.05).

C9orf72 mutant mice generated by CRISPR/Cas9develop autoimmunityWe noted that some noncoding regions within the deleted locus showeda degree of conservation that raised the possibility that their disruptioncould contribute to the phenotypes observed in KOMP and Neo-deletedanimals. To rule out this possibility, we used CRISPR/Cas9 technologyto induce deletion mutations in exon 4 of C9orf72 (Fig. 7A), an exon pres-ent in all transcribed isoforms of both human and murine orthologs.Next-generation sequencing of PCR amplicons spanning the target siterevealed disruption of C9orf72 exon 4 in 23 of 24 CRISPR/Cas9-targetedmice (Fig. 7B, fig. S14, and table S2). In 11 of 24 CRISPR/Cas9-targetedanimals, we identified only premature stop codon mutations and no wild-type sequences, suggesting that these mice were compound heterozygousloss-of-function mutants for C9orf72 (Fig. 7B, fig. S14, and table S2).

All CRISPR/Cas9-targetedmice survived to adulthood, yet as they aged,their propensity to die increased, with only 10 of 24 animals surviving be-yond 500 days of age (Fig. 7C). Splenomegaly was apparent in all mice (n =7 of 7) necropsied (Fig. 7, D and E), whereas protrusions in the neck areaindicative of enlarged cervical lymph nodes were apparent in 3 of24 animals (fig. S14). Analysis of blood from mutant mice at day 310revealed a significant reduction in hematocrit (Fig. 7F; **P < 0.01) and asignificant increase in anti-dsDNA antibody activity (Fig. 7G; *P < 0.05).

Because CRISPR/Cas9-targeted animals were mosaic for mutationsin C9orf72, we crossed F0 mice with C57BL/6 to isolate F1 progenyharboring one wild-type allele and one mutated allele (fig. S14). Wethen interbred F1 progeny containing a 38-nucleotide deletion inC9orf72 exon 4, predicted to generate a premature stop codon after183 amino acids, to generate animals that were wild-type (+/+), het-erozygous (+/del), and homozygous (del/del) for this particular muta-tion (Fig. 7H and fig. S14). We again found that C9orf72 del/del F2mutants displayed significantly elevated anti-dsDNA antibody activityby day 150 (Fig. 7I; **P < 0.01).

DISCUSSION

Here, we report that eliminating function of mouse C9orf72 predis-poses animals to fatal immune defects and autoimmunity. We observedthat these classic features of autoimmunity (24, 29) developed synchro-nously, well before the onset of premature mortality. Furthermore,heterozygous animals were more susceptible than control mice to thegeneration of a limited repertoire of autoantibodies and were at in-creased risk of early mortality. Chronic inflammation is a known con-tributor to most, if not all, of the causes of death that we observed in+/− and −/− animals. Notably, the pattern of inflammatory cytokineschronically up-regulated in −/− animals included several members ofthe IL-23/IL-17 immune axis, which plays a pathogenic role in rheuma-toid arthritis, psoriasis, Crohn disease, and multiple sclerosis (30).

Our findings led us to posit that C9orf72 serves an important func-tion in the hematopoietic system. This hypothesis was substantiated

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by transplant studies in which mutant bone marrow transferred auto-immune phenotypes to wild-type recipients and wild-type bone marrowsuppressed immune deregulation and significantly extended life span inmutant animals. However, although the phenotype of mutant animalsthat received wild-type marrow was improved relative to mutant micereceiving mutant bone marrow, their phenotype was still substantiallyworse than wild-type animals receiving wild-type bone marrow. Fail-ure of wild-type bone marrow to completely rescue mutant mice leavesopen the possibility that C9orf72 also functions in a radiation-resistant cellpopulation, such as microglia, which are peripheral antigen-presentingcells, the dysfunction of which could lead to improper priming of adap-tive immune responses. C9orf72 might also function in the thymic ep-ithelium, which is critical for the establishment of peripheral tolerance.Alternatively, T cells themselves are well-known modulators of immu-nity, and the incomplete replacement of this compartment after trans-plantation provides another potential explanation for the incompleterescue observed. Our findings, and those that indicate that T cellfunction becomes compromised in mice harboring mutations in ad-ditional ALS-implicated genes (19, 31), suggest that investigatingwhether C9orf72 functions in this cell type is warranted. Althoughwe observed an increase in the abundance of CD25+ CD4+ cells, whichare enriched for T regulatory cells, in the spleens of mutant animals,whether mutant T regulatory cells harbor functional deficits that con-tribute to the phenotypes that we observed remains to be determined.Elevation of T regulatory cells could represent a compensatory reac-tion to the reduced functionality of this cellular subset or a homeostaticresponse to persistent inflammation.

We note that our study does have certain limitations and leaves sev-eral questions unresolved. Although our studies demonstrate that bonemarrow cells play a role in the phenotypes that we observed and suggestthat C9orf72 functions in hematopoietic derivatives of the bone marrow,we cannot rule out the possibility that C9orf72 acts through marrow-resident mesenchymal cells. Additionally, we did not perform an ex-haustive census of transplanted animals to monitor the extent ofengraftment into solid tissues, including the brain. Therefore, we can-not address whether incomplete imposition or rescue of mutant pheno-types after transplant might be due to variable infiltration of cells intoa given solid organ (32) rather than due to the function of C9orf72 innonhematopoietic cell types. In the future, it will be important to resolvethe identity of the specific cell types in which C9orf72 functions to pre-serve a normal immune response. We previously showed that C9orf72was highly expressed in neuronal cell types that are most sensitive to celldeath in ALS and FTD but is more modestly expressed in microglia. De-spite the high level of C9orf72 expression in the neuronal lineage, the datawe report here suggest that this gene acts through bone marrow–derivedcells in which it is expressed. It remains to be determined whetherC9orf72 serves some nonessential function in neurons that express it.

Recently, haploinsufficiency for TBK1, a kinase functioning in IFNsignaling and selective autophagy (33), has been shown to cause ALS andFTD [reviewed in (34)]. Tbk1 −/−mice display an embryonic lethal phe-notype; however, deletion of this gene on an outbred background leads tomonocytosis, splenomegaly, and infiltration of immune cells into the skin,lung, liver, and kidneys (35). Cre-mediated ablation of Tbk1 in T cells re-sulted in their activation and overaccumulation in the spleen and lymphnodes (19). These phenotypic commonalities observed upon loss of Tbk1andC9orf72 are striking.Whether these gene products act in a commonpath-way requires further investigation; however, it is notable that at least onereport has suggested that C9ORF72 may also function in autophagy (11).

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Whether malfunctions in the immune system directly contribute todegenerative phenotypes in ALS patients remains unresolved (36, 37).Studies have identified infiltrating T cells and hyperactivation of residentimmune cells in the postmortem spinal cords of ALS patients (38, 39).Patients with a rapid course of disease were also found to have fewercirculating T regulatory cells than did individuals with slower disease pro-gression (40), and levels of the proinflammatory cytokines IL-17 andIL-23 were found to be elevated in plasma and cerebral spinal fluid fromat least one ALS cohort (41). Recently, a large epidemiologic study foundsubstantially higher rates of autoimmune history in patients eventuallydeveloping ALS (42). However, studies of autoantibodies in ALS patientshave been mixed, with some finding evidence of their presence, whereasothers have not (36, 37). The ever-improving understanding of the genet-ic contributors to ALS suggests that revisiting immune phenotypes inpatients who have been stratified by C9ORF72, TBK1, and other geno-types could now be warranted.

There are several other phenotypic studies of C9orf72-deficient mice(43, 44). These reports provide direct confirmation of our finding thatC9orf72 mutant animals develop splenomegaly and lymphadenopathy.Furthermore, Atanasio and colleagues found, as we report here, that mu-tant animals exhibited elevated plasma cytokines and autoantibodies (43).However, there were a number of differences between our findings andthese recent studies that warrant future resolution. Most notably, althoughboth we and O’Rourke et al. generated C9orf72mutant animals using thesame targeting vector generated by the KOMP Consortium, heterozygousand homozygous mutants in our colony displayed an increased risk ofmortality whereas mutants in their colony did not. This discrepancy sug-gests that either environmental factors or differences in genetic back-ground may influence the longevity of mice lacking C9orf72, as is thecase with other models of autoimmunity (29). Additionally, both Atanasioet al. and O’Rourke et al. found an increase in macrophage number inmutant animals and suggested that inflammatory phenotypes found inthese mice may be due to dysfunction in this or other phagocytic celltypes. Our conclusion that C9orf72 functions in bone marrow–derivedcells is consistent with this idea. However, we also observed a muchbroader proliferative disorder affecting both myeloid and lymphoidlineages, leaving open the question of whether this gene may act in addi-tional hematopoietic cell types. In the future, conditional ablation ofC9orf72 in specific blood lineages should address where precisely this genefunctions to suppress pathological inflammation.

Overall, our findings have implications for planned therapeutic in-terventions in ALS patients harboring the C9ORF72 repeat expansion.Regardless of whether loss of function in C9ORF72 plays a central rolein the development of ALS, our experiments do strongly suggest thattherapeutic efforts to reduce expression of the repeat expansion, suchas with antisense oligonucleotides (9), should be designed and carriedout with caution. If not, chronic depression of the limited quantity ofnormal gene product that still remains in these ALS patients couldoccur, potentially resulting in autoimmunity.

MATERIALS AND METHODS

Study designThe goal of this study was to characterize the consequences of C9orf72loss-of-function mutations in mice. The experimental design involvedlong-term survival studies paired with age-matched and genotype-matched histologic, cellular, and biofluidic analysis. Predefined end-

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point criteria included greater than 15% loss of body weight, signs ofdistressed breathing, enlarged abdomen, severe dermatitis, lymph nodesgreater than 15 mm that inhibited mobility, inability of the mouse to rightitself, or severe prolapse. Exploratory experiments were performed in ex-cess of five mice per genotype. Sample sizes for BMT studies werecalculated using G-power analysis based on previously defined effect sizes.Donor and recipient mice were randomized into treatment groups, and alloutliers were included in data analysis. All experimental protocols and pro-cedures were approved by the Animal Committee of Harvard University.

RNA extraction and quantitative PCRTotal RNA was isolated using TRIzol (Gibco). First-strand complementaryDNA was synthesized using iScript (Bio-Rad). C9orf72 transcript abun-dance was analyzed using SYBR Green on CFX96 Real-Time System(ABI). C9orf72 exon 6 to 8 primers are as follows: 5′-GCAGTGCAGAG-AAAGTAAATAAGATAG-3′ and 5′-ACTGCCTGTTGCATCCTTTAG-3′.

Fixation, sectioning, and tissue stainingMice were anesthetized with Avertin and perfused with phosphate-buffered saline and then with 4% paraformaldehyde (PFA). Tissueswere harvested and postfixed in 4% PFA overnight at 4°C. Tissuewas dehydrated, embedded in paraffin, sectioned at 6 mm, and stainedfor hematoxylin and eosin. Sections were examined in a blinded mannerand imaged using Zeiss AX10.

Western blottingTotal protein was extracted from a frozen brain with a reducing sam-ple buffer (radioimmunoprecipitation assay) containing complete in-hibitor cocktail (Roche). Protein (15 mg) was separated on 4 to 20%SDS–polyacrylamide gel, transferred to polyvinylidene difluoridemembrane, blocked with 5% bovine serum albumin, incubated withprimary antibodies [anti–C9ORF72–N-terminal (Proteintech), anti–C9ORF72–C-terminal (Abgent), and anti–a-tubulin (Abcam)] anddetected by enhanced chemiluminescence (GE Amersham).

Blood and cytokine measuresPeripheral blood was collected via mandible puncture into EDTA-coated tubes. Blood counts were assessed using a Hemavet (Abaxis)and then centrifuged to pellet cells, and plasma was harvested fromthe supernatant. Luminex-based multiplexed fluorescence assay wasused to assess 36 cytokines and chemokines.

Autoantibody profilingPlasma was diluted 1:200 and assessed using a mouse a-dsDNA total-Ig kit (Alpha Diagnostic International). Antibodies against 124 auto-antigens were measured on autoantigen arrays (26).

Spleen and lymph node fluorescence-activated cell sortingSpleens were mashed between two glass slides and passed repeatedlythrough a 25 5/8G needle to dissociate single cells. After RBC lysis, cellswere quantified using a Countess (Invitrogen). Lymph nodes were pro-cessed as above without RBC lysis. Cells were stained using the follow-ing antibodies (BioLegend): B220 (RA3-6B2), CD19 (6D5), k (RMK-45),l (RML-42), CD11c (N418), CD8a (53-6.7), CD3e (145-2C11), CD4(GK1.5), CD62L (MEL-14), CD44 (IM7), CD25 (PC61), Pan-NK (DX5),CD11b (M1/70), Ly6G (1A8), CD115 (AFS98), F4/80 (BM8), and 4′,6-diamidino-2-phenylindole (Sigma).

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Bone marrow transplantationFemale B6.SJL-PtprcaPepcb/BoyJ (The Jackson Laboratory) mice wereused as age-matched wild-type bone marrow donors and recipients. Fe-male KOMP C9orf72 −/− mice were used as age-matched bone marrowdonors and recipients. At D110, marrow from femur and tibias were har-vested from two wild-type and two −/− donors, flushed, single cell–dissociated, counted, pooled, and diluted to 5E6 cells/100 ml. Recipientanimals received two rounds of 550 rad separated by 3 hours. 5E6 bonemarrow cells were injected into the tail vein. The recipients were main-tained on pH 3.0 water for 2 weeks. Reconstitution efficiency in RBC-lysed peripheral blood at 17 weeks after transplant was measured usingthe following antibodies (BioLegend): CD45.2 (104), CD3e (145-2C11),B220 (RA3-6B2), Ly6G (1A8), CD11b (M1/70), and CD45.1 (A20).

CRISPR/Cas9 sequencingTail DNA was PCR-amplified with primers containing flanking adap-ters, followed by PCR barcoding with 24 unique barcodes. Sampleswere pooled, cleaned (Promega Wizard DNA clean-up kit), sequencedvia MiSeq, and analyzed (45).

StatisticsAll statistical calculations were performed using GraphPad Prism.Tests between two groups used two-tailed Student’s t test. Tests be-tween multiple groups used one-way analysis of variance (ANOVA)with Tukey multiple comparisons. Tests between multiple groups overtime used two-way ANOVA with Dunnett’s multiple comparisons.Survival curves were evaluated by generalized Wilcoxon.

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SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/347/347ra93/DC1TextFig. S1. Validation of C9orf72 loss-of-function allele.Fig. S2. Analysis of spinal motor neurons.Fig. S3. Motor cortex histology.Fig. S4. Histology of thalamus, CA1, and cerebellum.Fig. S5. GFAP expression in the central nervous system.Fig. S6. Individual mouse weight curves after disease onset.Fig. S7. Splenocyte-gating scheme.Fig. S8. Bone marrow histology and cell counts.Fig. S9. Analysis of B and T cells in cervical lymph node.Fig. S10. Hematopoietic liver infiltrates occur in a subset of mutant animals.Fig. S11. Analysis of cytokines and chemokines.Fig. S12. Analysis of CD4+ CD25+ cells.Fig. S13. Cellular and organismal phenotypes after bone marrow transplant.Fig. S14. CRISPR/Cas9-targeted mutations in exon 4 of C9orf72.Table S1. Analysis of autoantibodies in Neo-deleted mice.Table S2. CRISPR/Cas9-induced mutations in C9orf72 exon 4.Video 1. End-stage −/− marrow donor to −/− recipient.Video 2. End-stage −/− marrow donor to WT recipient.Video 3. End-stage WT marrow donor to −/− recipient.

REFERENCES AND NOTES

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4. S. Byrne, M. Elamin, P. Bede, A. Shatunov, C. Walsh, B. Corr, M. Heverin, N. Jordan, K. Kenna,C. Lynch, R. L. McLaughlin, P. M. Iyer, C. O’Brien, J. Phukan, B. Wynne, A. L. Bokde, D. G. Bradley,N. Pender, A. Al-Chalabi, O. Hardiman, Cognitive and clinical characteristics of patients withamyotrophic lateral sclerosis carrying a C9orf72 repeat expansion: A population-based cohortstudy. Lancet Neurol. 11, 232–240 (2012).

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Acknowledgments: We thank the Eggan laboratory, the HSCRB (Harvard Department of StemCell and Regenerative Biology) Histology Core, and M. Charlton, A. Wagers, D. Scadden, andK. Hochedlinger for advice, technical support, and manuscript review. Funding: K.E. wassupported by the Howard Hughes Medical Institute, p2ALS, Target ALS, and NIH5R01NS089742.F.T.M. was supported by the NIH (5K99NS083713), the Wellcome Trust, the Academy of MedicalSciences, and the Medical Research Council (MR/P501967/1). Author contributions: K.E. conceivedthe project; K.E., A.B., and N.S. designed the experimental plan. A.B., N.S., J.-Y.W., A.S., K.K., and S.S.-U.supported animal experiments. R.M. supported DNA sequence analysis. D.A.M. evaluated staining.S.G. and Q.-Z.L. performed autoantibody array. A.S., M.H.S., J.J.T., D.J.R., L.Z., and L.D.N. performedhematological analysis. A.B. and F.T.M. performed CRISPR/Cas9 design. A.B., N.S., R.M., and K.E. wrotethe manuscript. K.E. supervised the project. Competing interests: The authors declare that they haveno competing financial interests. Data and materials availability: Whole-genome sequencingdata for this study have been deposited in Sequence Read Archive SRP073407.

Submitted 3 March 2016Accepted 21 June 2016Published 13 July 201610.1126/scitranslmed.aaf6038

Citation: A. Burberry, N. Suzuki, J.-Y.Wang, R.Moccia,D. A.Mordes,M.H. Stewart, S. Suzuki-Uematsu,S. Ghosh, A. Singh, F. T.Merkle, K. Koszka, Q.-Z. Li, L. Zon, D. J. Rossi, J. J. Trowbridge, L. D. Notarangelo,K. Eggan, Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmunedisease. Sci. Transl. Med. 8, 347ra93 (2016).

Ja


nuary 14, 2021


disease mouse ortholog cause fatal autoimmuneC9ORF72Loss-of-function mutations in the

J. Rossi, Jennifer J. Trowbridge, Luigi D. Notarangelo and Kevin EgganSuzuki-Uematsu, Sulagna Ghosh, Ajay Singh, Florian T. Merkle, Kathryn Koszka, Quan-Zhen Li, Leonard Zon, Derrick Aaron Burberry, Naoki Suzuki, Jin-Yuan Wang, Rob Moccia, Daniel A. Mordes, Morag H. Stewart, Satomi

DOI: 10.1126/scitranslmed.aaf6038, 347ra93347ra93.8Sci Transl Med

investigations are warranted into whether disruptions in immunity contribute to disease in patients. acts through hematopoietic cells to maintain normal immune function and suggest thatC9orf72conclude that

transplantation of mutant bone marrow into normal animals was sufficient to cause autoimmunity. The authorsfound that transplantation of normal bone marrow into mutant animals ameliorated this phenotype, whereas

cause mice to develop features of autoimmunity. They furtherC9orf72mutations disrupting the normal function of . demonstrate thatet aldementia, yet the function of this gene is still poorly defined. In new work, Burberry

are a common contributor to amyotrophic lateral sclerosis and frontotemporalC9ORF72Mutations in C9orf72, a suppressor of autoimmunity?

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