Case Report J Vet Intern Med 2017;31:539–544
Precision Medicine in Cats: Novel Niemann-Pick Type C1 Diagnosed by Whole-Genome Sequencing D.A. Mauler, B. Gandolfi, C.R. Reinero, D.P. O’Brien, J.L. Spooner, L.A. Lyons, and 99 Lives Consortium State-of-the-art health care includes genome sequencing of the patient to identify genetic variants that contribute to either the cause of their malady or variants that can be targeted to improve treatment. The goal was to introduce state-of-the-art health care to cats using genomics and a precision medicine approach. To test the feasibility of a precision medicine approach in domestic cats, a single cat that presented to the University of Missouri, Veterinary Health Center with an undiagnosed neurologic disease was whole-genome sequenced. The DNA variants from the cat were compared to the DNA variant database produced by the 99 Lives Cat Genome Sequencing Consortium. Approximately 259 genomic coverage was produced for the cat. A predicted p.H441P missense mutation was identified in NPC1, the gene causing Niemann-Pick type C1 on cat chromosome D3.47456793 caused by an adenine-to-cytosine transversion, c.1322A>C. The cat was hom*ozygous for the variant. The variant was not identified in any other 73 domestic and 9 wild felids in the sequence database or 190 additionally genotyped cats of various breeds. The successful effort suggested precision medicine is feasible for cats and other undiagnosed cats may benefit from a genomic analysis approach. The 99 Lives DNA variant database was sufficient but would benefit from additional cat sequences. Other cats with the mutation may be identified and could be introduced as a new biomedical model for NPC1. A genetic test could eliminate the disease variant from the population. Key words: Feline; Felis silvestris catus; Lysosomal storage; NPC1; WGS.
T
he genetic and genomic resources available for health studies of the domestic cat are becoming
From the Department of Veterinary Medicine & Surgery, College of Veterinary Medicine, University of Missouri – Columbia, Columbia, MO (Mauler, Gandolfi, Reinero, O’Brien, Lyons); Klingele Veterinary Clinic, Quincy, IL (Spooner). Work: The cat was referred to the Veterinary Health Center at the University of Missouri, Columbia, Missouri, the genetic studies and analysis at the University of Missouri, Columbia, Missouri, the whole-genome sequencing was performed as fee-for-service at The McDonnell Genome Institute at Washington University in St. Louis, Missouri, and partial genome sequence analysis was conducted as fee-for-service at Maverix Biomics, Inc., San Mateo, California. Funding: This work was supported in part by funding from the National Center for Research Resources R24 RR016094 and the Office of Research Infrastructure Programs/OD R24OD010928, and the Winn Feline Foundation (W10-014, W16-030) the George Sydney and Phyllis Redman Miller Trust (MT-13-010), the National Geographic Society Education Foundation (2P-14), public and private donations to the 99 Lives Cat Genome Initiative project, including the Associazione Nazionale Felina Italiana, Zoetis, Orivet Genetic Pet Care, Langford Veterinary Services, the World Cat Federation, public donations, and the University of Missouri, College of Veterinary Medicine Gilbreath McLorn endowment. Presentation: This paper has not been presented at any scientific meeting. Authors contributed equally to the research. Corresponding author: L.A. Lyons, PhD, Department of Veterinary Medicine & Surgery, College of Veterinary Medicine, University of Missouri – Columbia, Columbia, MO 65211; e-mail: [emailprotected].
Submitted June 2, 2016; Revised August 4, 2016; Accepted September 19, 2016. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. DOI: 10.1111/jvim.14599
Abbreviations: ALP ALT bp bpm KIT MRI NPC1 NPC2 NPC OMIA OMIM PCR qPCR WGS
alkaline phosphatase alanine aminotransferase base pair beats per minute v-kit Hardy-Zuckerman 4 feline sarcoma oncogene hom*olog magnetic resonance imaging Niemann-Pick type C1 Niemann-Pick type C2 Niemann-Pick type C Online Mendelian Inheritance in Animals Online Mendelian Inheritance in Man polymerase chain reaction quantitative PCR whole-genome sequencing
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robust and cost efficient. The sequencing of a cat’s entire genome can now be completed for about the cost of a magnetic resonance imaging (MRI) scan. In humans, rapid turnaround whole-genome sequencing such as the 26-hour genome efforts has demonstrated how genomic medicine can be applied to health management for acute care cats with time-critical morbidity and mortalities.1,2 Although the availability of the bioinformatics infrastructure and turnaround time are not yet as accessible in cats as for humans, the DNA variant database developed by the 99 Lives Cat Genome Sequencing Initiative has proven a valuable first step. Developed from a variety of cats comprising diverse populations and breeds, including those with and without known genetic health problems, the cat variant database supports the identification of DNA variants that are causal for health conditions suspected to have a genetic component. Two whole-genome sequencing (WGS) studies have already identified a
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DNA variant in cats associated with progressive retinal atrophy in Persians, a variant causing the bobbed tail of the Japanese Bobtail breed,3 and one responsible for congenital myasthenic syndrome in Devon rex and Sphynx—related cats.4 Precision medicine is an emerging approach for disease diagnosis, treatment, and prevention that takes into account individual variability in genes, environment, and lifestyle. Most medical treatments have been developed for the “average patient”. Precision medicine gives clinicians tools to better understand the complex mechanisms underlying a cat’s health, disease, or condition, and to better predict which treatments will be most effective, or to determine polymorphisms requiring different drug dosages (pharmaco*kinetics).5 Overall, an individual’s specific genetic makeup will become an intricate part of their standard health care. This case report shows the potential to apply precision medicine to the diagnosis of neurologic disease in cats. A 12-week-old silver tabby intact female cat of suspected American shorthair lineage presented for a new kitten examination. Abnormal physical examination findings included a pendulous abdomen, soft nonformed feces, and a shaky, unsteady, hypermetric gait causing the cat to fall over. A presumptive diagnosis of cerebellar hypoplasia was made. Two months later, she presented for ovariohysterectomy. During the presurgery examination, her neurologic signs had progressed and she was having difficulty ambulating. Although well hydrated, her weight gain was 0.05 kg in 4 weeks. Presurgery blood tests revealed hypoalbuminemia (1.7 g/ dL, reference interval, 2.5–4.4 g/dL), elevated ALT (436 U/L, reference interval 10–118 U/L) and ALP activities (184 U/L, reference interval 20–150 U/L), hyperbilirubinemia (0.6 mg/dL, reference interval 0.1–0.6 mg/dL), and hyperphosphatemia (7.0 mg/dL, reference interval 2.9–6.6 mg/dL). Abdominal radiographs and ultrasonography showed generalized hepatomegaly and splenomegaly. The hepatic parenchyma was hypoechoic and hom*ogenous. The hepatic vasculature was within normal limits. The gall bladder was mildly distended with anechoic bile. Although enlarged, the spleen was hom*ogenous in echogenicity. The remainder of the abdominal ultrasound examination was unremarkable. Postprandial bile acids were elevated (42.1 lmol/L, reference interval C. The cat of this report was hom*ozygous for the variant, and no other cats in the dataset had the variant. The histidine at position 441 is highly conserved across species (Fig 1). PolyPhen-2 predicts the amino acid change to be benign with a damaging score of 0.43 (sensitivity: 0.89; specificity: 0.90).8 The NPC1 variant is rare as it was not identified in 190 cats (Figure S1).
Discussion Niemann-Pick type C (NPC) disease (NP-C; OMIM#257220 and OMIM#607625) is an autosomal recessive neurovisceral lysosomal storage disorder that results in defective intracellular transport of cholesterol. Greater than 95% of all human NPC cases are because of variants in the NPC1 gene mapped to chromosome 18q11,9 which is hom*ologous to cat
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chromosome D3.7,10 NPC1 in cats was first described as a lipid disorder in a Siamese.11–15 A feline model of NPC1 (OMIA 000725-9685) is characterized and is phenotypically, morphologically, and biochemically similar to human NPC1.16–22 Affected cats show cerebellar and vestibular signs beginning as early as 6 weeks of age. The ataxia progresses to the point where cats fell frequently at 11.8 1.9 weeks of age and were unable to take 1 step without falling at 17 2.1 weeks age. By 19 3 weeks of age, they were unable to stand without assistance, and they were euthanized at 20.5 4.8 weeks of age (range 11–29 weeks).21 The cat in this study was not evaluated before 12 weeks of age but had clear ataxia by 12 weeks of age with difficulty walking by 20 weeks of age. When examined at 24 weeks of age, the cat could not walk without assistance and she was euthanized at 38 weeks of age. Thus, the onset and progression of neurologic signs in this cat are comparable to those observe in the feline NPC1 colony. The cat in this report survived longer before euthanasia but that likely reflects additional nursing care provided to a pet versus cats in a research colony and reluctance to euthanize on the part of the owner. Serum ALP and ALT activities were elevated, and albumin concentrations decreased in the cat in this report comparable to NPC1 colony cats.22 Histopathology and biochemical studies would be necessary to confirm the diagnosis of NPC1 and demonstrate an effect of the variant identified on function. Unfortunately, the owners declined permission for biopsy or necropsy. Thus, we cannot prove the diagnosis of NPC1, which is a limitation of this study. Complementation studies using cultured fibroblasts from NPC-affected cats and NPC1-affected humans support that the gene responsible for the NPC phenotype in this colony of cats is orthologous to the gene responsible for the major form of human NPC, NPC1.23 A single base substitution (c.2864G>C) is identified in these NPC1-affected cats and in silico predicts a cysteine to a serine change (p.C955S)24 (GenBank # AF503633 and AF503634). The known cat NPC1 variant is predicted to be probably damaging using PolyPhen-2 with a score of 0.981 (sensitivity: 0.75; specificity: 0.96).8 Heterozygous cats also have metabolic manifestations.19 The parents of the cat could not be obtained for evaluation. In addition, a published intronic c.82+5G>A mutation, which leads to a splicing error in another gene associated with NPC in humans
Fig 1. Protein alignment of NPC1 in cats and other species within critical region for the cat variant H441P. The mutation is within the luminal topological domain that includes amino acids 372 to 620. The conserved variant site for this case (p.H441P) is presented in bold italics and underlined. NPC1 in Felis catus has two amino acids shorter compared to human. In humans, three known variants at codon positions 433, 434, and 451 are associated with a disease phenotype and are bold in the alignment.33–35
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(NPC2), has been identified in cats with similar presentation,25 but no significant variants were identified in NPC2 in the cat of this report. However, as presented in Orphanet (http://www.orpha.net/consor/cgibin/index.php?lng=EN), the mutations in NPC1 occur in 95% of the families with NPC type disease and more than 230 mutations have been identified. The identified NPC1 variant in this cat is novel and predicts a nonconservative missense mutation, a histidine-to-proline substitution at position 441 of the NCP1 feline protein. The histidine is highly conserved in several species, and prolines generally cause significant disruption to the normal protein structure. The newly identified variant is not known in human patients, but several proline changes and a terminal variant are within the region of codons 421–474 (http:// www.hgmd.cf.ac.uk/ac).26 Although the PolyPhen-2 prediction was suggested as a benign effect on the protein, other benign predictions are associated with phenotypic effects in cats, such as the Gloving variants associated with KIT that are common to Birmans.7 The NPC1 variant is rare as it was not identified in 190 cats or the other 73 domestic cats and 9 wild felids in the 99 Lives sequence database. However, the population of origin of the affected cat could not be established for further screening of a specific breed. Precision medicine by WGS has proven to be useful for diagnosis and rapid intervention and treatment for critically ill children.1,2 Although the treatments for NPC1 are not ready for clinical trials in cats and would not have assisted the present cat, other undiagnosed diseases in cats could benefit from genomics techniques. A second NPC1 biomedical model in cats could be established if other afflicted or carrier cats could be identified, which would further support the development of potential treatments for the disease. Currently, cyclodextrin and miglustat have shown improvement in Purkinje cell survival, thereby preventing cerebellar dysfunction in cats.27–30 Assuming the cat of this report is representative of a specific breed, its NPC1 variant could be segregating in a breeding population and other hom*ozygous recessive cats may be produced which will succumb to the same condition if a genetic test is not implemented to avoid matings between carriers. To date, over 40 genes with approximately 70 DNA variants have been documented to cause phenotypic, disease, or blood type variations in the domestic cat (for review).31 The clinical descriptions and phenotypes of each of these diseases and traits have been curated at the Online Mendelian Inheritance in Animals (OMIA) website (http://omia.angis.org.au/home/), which is an invaluable resource comparison of the phenotypes across 2216 animal species.32 These known and any newly identified DNA variants can be genotyped rapidly and cost-effectively in panels appropriate for breeds, populations, or in cats as part of wellness care. The vigilance of veterinarians and their collaboration with geneticists could lead to the rapid discovery of undiagnosed genetic conditions in cats, which lead to more effective and proactive treatments and preventative strategies. Whole-genome sequencing of rare and
undiagnosed feline cases may be resolved using the precision medicine approach.
Acknowledgments We appreciate the laboratory assistance of Nick Gustafson and Erica Creighton. We appreciate the provision of cat DNA samples by Cristy Bird, Sam Boutin, Bruno Chomel, Jeanette Coleman-Hall, Johnny Gobble, Terri Harris, Anthony Hutcherson, Kyung Sik Kim, Mark Kantrowitz, Sheri Moreau, Nassem N. Naimi of Best Friend Veterinary Clinic in Amman, Jordan, Anthony Nichols, Jean Papo, Julie Pomerantz, John Snape, Susanne and Claus Wehnert, Nancy Carpenter at Utah’s Hogle Zoo, Ashleigh Lutz-Nelson at San Francisco Zoo & Gardens, and Julie Feinstein at the American Museum of Natural History, Franklin Whittenberg. Conflict of Interest Declaration: Dr. Lyons and Dr. Gandolfi have received funds from the Veterinary Genetics Laboratory (VGL) at the University of California, Davis. This laboratory could develop a commercial service for this mutation and offer genotyping to the public and scientific community. Part of the VGL’s income could be used to support additional research for Drs. Lyons and Gandolfi. Off-label Antimicrobial Declaration: Authors declare no off-label use of antimicrobials.
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Appendix 1. 99 Lives Consortium (83 cat analysis) Leslie A. Lyons1, Danielle Aberdein2, Paulo C. Alves3,4, Gregory S. Barsh5,6, Holly C. Beale7, Adam R. Boyko8, Jeffrey A. Brockman9, Marta G. Castelhano10, Patricia P. Chan7, N. Matthew Ellinwood11, Jonathan E. Fogle12, Dorian J. Garrick2,11, Christopher R. onen14, Maria Kaukonen14, Helps13, Marjo K. Hyt€ Christopher B. Kaelin5,6, Emilie Leclerc15, Tosso Leeb16, Hannes Lohi14, Maria Longeri17, Richard Malik18, Michael J. Montague19, John S. Munday2, William J. Murphy20, Niels C. Pedersen21, Max F. Rothschild11, Joshua A. Stern21, William F. Swanson22, Karen A. Terio23, Rory J. Todhunter10, Yu Ueda21, Wesley C. Warren19, Elizabeth A. Wilcox10, Julia H. Wildschutte24, Barbara Gandolfi1. 1 Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, 65211. 2 Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North 4474 New Zealand. 3 CIBIO/InBIO, Centro de Investigacß~ ao em Biodiversidade e Recursos Geneticos/InBIO Associate Lab & Faculdade de Ci^encias, Universidade do Porto, Campus e Vair~ ao, 4485–661 Vila do Conde, Portugal. 4 Wildlife Biology Program, University of Montana, Missoula, Montana, 59812. 5 HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, 35806. 6 Department of Genetics, Stanford University, Stanford, California, 94305. 7 Maverix Biomics, Inc., San Mateo, California, 94402. 8 Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, 14853. 9 Hill’s Pet Nutrition Inc., PO Box 1658, Topeka, KS 66601. 10 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, 14853. 11 Department of Animal Science, College of Agriculture and Life Sciences, Iowa State University, Ames, Iowa, 50011. 12 College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607. 13 Langford Veterinary Services, University of Bristol, Langford, Bristol, BS40 5DU UK.
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14 Department of Veterinary Biosciences and Research Programs Unit, Molecular Neurology, University of Helsinki and Folkh€ alsan Research Center, Helsinki 00014 Finland. 15 Diana Pet food, Inc. SPF – ZA du Gohelis, 56250 Elven, France. 16 Vetsuisse Faculty, Institute of Genetics, University of Bern, 3001 Bern, Switzerland. 17 Dipartimento di Medicina Veterinaria, University of Milan, 20122 Milan, Italy. 18 Centre for Veterinary Education, University of Sydney, Sydney, NSW, 2006 Australia. 19 The McDonnell Genome Institute, Washington University School of Medicine, St. Louis, Missouri, 63108. 20 Department of Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, Texas, 77845. 21 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California at Davis, Davis, California, 95616.
22 Center for Conservation and Research of Endangered Wildlife (CREW), Cincinnati Zoo & Botanical Garden, Cincinnati, Ohio, 45220. 23 Zoological Pathology Program, University of Illinois, Brookfield, IL 60513. 24 Bowling Green State University, Department of Biological Sciences, Bowling Green, OH 43403.
Supporting Information Additional Supporting Information may be found online in the supporting information tab for this article: Table S1. Genotyping primer sequences for cat NPC1. Figure S1. Cartesian plot of genotypes detected by mass spectrometry genotyping. Plot representing the genotyping results of 96 cats, only the affected NiemannPick type C cat (upward triangle) was hom*ozygous for the identified variant (c.1322A>C). One sample (circle) was excluded for low genotype quality.
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