Variants within the SP110 nuclear body protein modify PNAS PLUS risk of canine degenerative myelopathy Emma L. Ivanssona,b,1,2, Kate Megquiera,b, Sergey V. Kozyreva, Eva Muréna, Izabella Baranowska Körbergc,3, Ross Swoffordb, Michele Koltookianb, Noriko Tonomurab,d, Rong Zenge, Ana L. Kolicheskie, Liz Hansene, Martin L. Katzf, Gayle C. Johnsone, Gary S. Johnsone, Joan R. Coatesg, and Kerstin Lindblad-Toha,b,1 aScience for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, 751 23 Uppsala, Sweden; bBroad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA 02142; cDepartment of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, 750 07 Uppsala, Sweden; dDepartment of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA 01536; eDepartment of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211; fMason Eye Institute, School of Medicine, University of Missouri, Columbia, MO 65201; and gDepartment of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211 Edited by Stephen T. Warren, Emory University School of Medicine, Atlanta, GA, and approved April 15, 2016 (received for review January 7, 2016) Canine degenerative myelopathy (DM) is a naturally occurring several clinical subtypes with variable phenotypic presentation and neurodegenerative disease with similarities to some forms of prognosis. amyotrophic lateral sclerosis (ALS). Most dogs that develop DM Over 20 y ago, a mutation in the superoxide dismutase 1 gene are homozygous for a common superoxide dismutase 1 gene (SOD1) was the first genetic risk factor to be identified (4). To (SOD1) mutation. However, not all dogs homozygous for this date, more than 160 SOD1 mutations involving all five exons mutation develop disease. We performed a genome-wide asso- have been identified in patients with ALS (alsod.iop.kcl.ac.uk/) ciation analysis in the Pembroke Welsh Corgi (PWC) breed com- (5). SOD1 has been followed by a growing list of ALS-associated paring DM-affected and -unaffected dogs homozygous for the – SOD1 genes (6 8), including an intronic repeat expansion in chromo- mutation. The analysis revealed a modifier locus on canine C9ORF72 chromosome 25. A haplotype within the SP110 nuclear body pro- some 9 open reading frame 72 ( ) present in patients SP110 tein ( ) was present in 40% of affected compared with 4% of Significance unaffected dogs (P = 1.5 × 10−5), and was associated with in- creased probability of developing DM (P = 4.8 × 10−6) and earlier − onset of disease (P = 1.7 × 10 5). SP110 is a nuclear body protein Degenerative myelopathy (DM) is a canine disease very similar involved in the regulation of gene transcription. Our findings to amyotrophic lateral sclerosis (ALS) in humans. We previously suggest that variations in SP110-mediated gene transcription showed that DM is a promising model for ALS, because genome- may underlie, at least in part, the variability in risk for developing wide association identified a mutation in superoxide dismutase SOD1 DM among PWCs that are homozygous for the disease-related 1gene( ), a known ALS gene. This mutation found in many SOD1 mutation. Further studies are warranted to clarify the ef- dog breeds increases the risk of DM, and the pathological find- fect of this modifier across dog breeds. ings and clinical progression of the two diseases are similar. In this study, we identify a modifier gene, SP110 nuclear body protein (SP110), which strongly affects overall disease risk and degenerative myelopathy | amyotrophic lateral sclerosis | ALS | SOD1 | SP110 age of onset in Pembroke Welsh Corgis at risk for DM. Dissecting the complex genetics of this disease in a model organism may myotrophic lateral sclerosis (ALS) is the most common lead to new insights about risk and progression in both canine Aadult-onset motor neuron disorder, with 50% of patients and human patients. dying within 2–3 y of the onset of clinical signs (1). Despite sig- nificant progress in the mapping of genetic risk loci, development Author contributions: E.L.I. and K.L.-T. designed research; E.L.I., K.M., S.V.K., E.M., I.B.K., of successful therapeutic strategies has remained elusive, in part, R.S., M.K., N.T., R.Z., A.L.K., L.H., M.L.K., G.C.J., G.S.J., J.R.C., and K.L.-T. performed re- due to the heterogeneity of the disease both genetically and phe- search; E.L.I., K.M., and S.V.K. analyzed data; and E.L.I. and K.M. wrote the paper. notypically. Further genetic dissection will facilitate the discovery Conflict of interest statement: A DNA test to identify dogs at risk of developing degen- of modifier genes, which influence disease onset and severity, and erative myelopathy is the subject of four awarded patents (European Patent 2247752, Australian Patent 2009212473, Japanese Patent 5584916, and Mexico Patent 326951) and may point the way to new therapeutic approaches. In this study, one pending patent application (Canada Patent 2,714,393). Three of the coauthors (G.S.J., we detail the use of a comparative approach to identify a genetic J.R.C., and K.L.-T.) are co-inventors listed on these patents and patent applications. A modifier that affects disease penetrance and age of onset in de- patent application was filed as to certain subject matter of this manuscript. GENETICS generative myelopathy (DM), a canine model of ALS. This article is a PNAS Direct Submission. The dog is a particularly powerful comparative disease model for Freely available online through the PNAS open access option. genetic studies of complex traits, combining aspects of the tracta- Data deposition: The National Center for Biotechnology Information (NCBI) Gene Expres- bility of a model organism with the advantages of genetic trait sion Omnibus (GEO) www.ncbi.nlm.nih.gov/geo accession numbers for the genome-wide mapping in population isolates, enabling the mapping of genetic risk association data presented in this paper are GSE80735 (PWC) and GSE80315 (Boxer). The NCBI Single Nucleotide Polymorphism Database (dbSNP) accession numbers for the Illu- factors using modest sample sizes (2). Dogs are predisposed to mina MiSeq-detected variants reported in this paper are 1987230493–1987230525. The many of the same complex diseases that humans are, share an en- NCBI Sequence Read Archive (SRA) accession numbers for the whole-genome sequences vironment with their human owners, and receive a sophisticated of three PWCs reported in this paper are SRX745862–SRX745864. The GenBank accession level of medical surveillance and care (3). numbers for the canine SP110 alternative transcripts reported in this paper are KP245899– ALS and canine DM are similar at a phenotypic, clinical, and KP245902. 1 genetic level. ALS is characterized by progressive loss of motor To whom correspondence may be addressed. Email: [email protected] or kersli@ broadinstitute.org. function and is characterized by stiffness and slowing of movements, 2Present address: Department of Medical Epidemiology and Biostatistics, Karolinska In- difficulty in speaking and swallowing, muscle atrophy, and severe stitutet, 171 77, Stockholm, Sweden. weakness culminating in paralysis. Mortality is typically secondary to 3Present address: Department of Women’s and Children’s Health, Karolinska Institutet, failure of the respiratory muscles. Familial forms of the disease ac- Karolinska University Hospital Solna, 171 76, Stockholm, Sweden. – – count for 5 10% of cases; the most common age of onset is 47 52 y This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. for familial ALS and 58–63 y for sporadic disease (1). There are 1073/pnas.1600084113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1600084113 PNAS | Published online May 16, 2016 | E3091–E3100 Downloaded by guest on September 24, 2021 with sporadic ALS (9, 10) and 38% of patients with familial ALS and changes in the gene isoform ratio of SP110 that may be relevant (11). A recent study reported that some patients with familial to disease development. ALS harbor mutations in more than one of the recognized ALS genes, including SOD1, C9ORF72, TARDBP, fused in sarcoma Results (FUS), and ANG (12). A large-scale exome sequencing study GWA Analysis Detects a Modifier Locus on cfa25. We performed a identified TBK1 as an ALS susceptibility gene (13). The same GWA analysis in at-risk PWC dogs homozygous for the SOD1 genes associated with familial ALS have been found to harbor risk allele to detect genetic modifier loci that differentiate be- mutations in patients with sporadic ALS (7, 8). In summary, the tween dogs that developed the disease early and those dogs that current knowledge supports genetic heterogeneity in ALS etiology did not develop disease even at an advanced age. By comparing and suggests that genetic factors may play a role in patients with cases with a confirmed diagnosis and early onset of DM signs apparently sporadic disease. with older dogs without any signs of the disease, we obtained Like ALS, canine DM is a naturally occurring, progressive phenotypes that were clearly defined and well separated. GWA adult-onset disease that leads to paralysis and death (14). The analysis was performed in a final dataset of 15 affected and 31 first clinical signs usually occur after 7 y of age and include general unaffected PWCs. Quality control left 119,768 SNPs at a total proprioceptive ataxia and asymmetrical spastic weakness of the hind genotyping rate of 99.9% for analysis. There were no outliers in limbs. Signs then progress to paraplegia, thoracic limb weakness, the dataset according to the multidimensional scaling plot (SI and, ultimately, flaccid tetraplegia (15). A presumptive clinical Appendix, Fig. S1) and the lambda (genomic inflation factor) was diagnosis is made by ruling out potential causes of compressive 0.99, indicating successful control of population stratification.