A novel missense mutation of the NAT10 gene in a juvenile Schnauzer dog with chronic respiratory tract infections

Abstract An 18‐month‐old intact male Schnauzer dog was evaluated for chronic, lifelong respiratory tract infections that were unresponsive to administration of a variety of antibiotics and corticosteroids. The dog developed persistent vomiting and diarrhea around 1 year of age that was minimally responsive to diet change, antibiotics, and corticosteroids. Despite supportive care, the dog was ultimately euthanized at 20 months of age due to persistent respiratory and gastrointestinal disease. Whole genome sequencing discovered a deleterious missense A/C mutation within the NAT10 gene, a gene essential for microtubule acetylation, appropriate ciliary development, and cytokinesis. Pipeline analysis of the genomes of 579 dogs from 55 breeds did not detect this mutation. Though never described in veterinary medicine, NAT10 mutation occurs in humans with ciliary aplasia, suggesting a pathophysiological mechanism for this dog and highlighting an associated mutation or possible novel genetic cause of chronic respiratory infections in dogs.


| CASE DESCRIPTION
An 18-month-old intact male Schnauzer dog was presented to the North Carolina State University Veterinary Hospital for evaluation of persistent cough and tachypnea. He was adopted from a breeder at 2 months of age, at which time he was reported as being clinically healthy. Soon after adoption, he developed a cough and was treated with a 4-week course of doxycycline by his primary veterinarian for presumptive canine infectious respiratory disease complex. His cough improved mildly, though it never fully resolved. By 7 months of age the dog was again presented to his primary veterinarian for persistent coughing and tachypnea. Thoracic radiographs demonstrated a diffuse bronchointerstitial pattern, and over the following months he was intermittently prescribed amoxicillin/clavulanic acid, prednisone, cefpodoxime, and chloramphenicol, all of which had a minimal effect on improving his clinical signs. At 10 months of age, a complete blood count (CBC) was performed, revealing a leukocytosis characterized by a moderate mature neutrophilia. At  Upon presentation to our institution 6 months later, the dog was abnormally small in stature, weighing 3.7 kg and having a body condition score of 2/9. Vital signs were unremarkable aside from constant tachypnea (respiratory rate 100 breaths/minute). Mild serous nasal discharge was noted bilaterally, and occasional crackles were heard confirmed that all subsets of lymphocytes were present in addition to marked granulocytosis. Despite its insensitivity for diagnosis of mucosal IgA deficiency, serum IgA was measured and found to be mildly decreased at 27 mg/dL (RI 35-270 mg/dL). Because of the dog's chronic respiratory infections that had been present from a very young age, ciliary dyskinesia was considered a possible differential diagnosis and a semen sample was attempted to be collected to evaluate the dog's sperm motility. Sample collection was unsuccessful, and so a 2 mL blood sample was collected for genomic DNA evaluation that could aid in the diagnosis of heritable diseases including neutrophil adhesion defects, ciliary dyskinesia, or immunoglobulin deficiency. The dog was discharged with azithromycin and recommendations for continued supportive care. Despite additional courses of antibiotics, the dog's respiratory status progressed to dyspnea, and due to a poor quality of life, he was ultimately euthanized at 20 months of age with no necropsy performed.
Genomic DNA was extracted using the QIAamp DNA Blood Kit standard protocol (Qiagen, Germantown, Maryland) and submitted for whole genome sequencing (WGS). DNA was processed for library preparation and whole genome sequencing using a 150 base pair (bp) paired-end read configuration on an Illumina HiSeq 4000 highthroughput sequencing system (Genewiz LLC, South Plainfield, New Jersey). Variant calling from next-generation sequencing data was performed using a standardized bioinformatics pipeline for all samples as described previously. 1

| DISCUSSION
In the dog reported here a novel deleterious variant was identified in the NAT10 gene that was predicted to create a deleterious change in the protein's structure. The NAT10 (N-acetyltransferase 10) protein is highly expressed in all human tissues, whereas in dogs it is highly expressed in many tissues including the lungs, brain, heart, and reproductive and gastrointestinal tracts. 8,9 NAT10 is a lysine acetyltransferase that acts on microtubules and histones, and is required for 18S rRNA biogenesis, nucleolar architecture, cytokinesis, and mitosis. [10][11][12] The structural change in the protein predicted here Although still in its relative infancy in veterinary medicine, whole genome sequencing has proven to be invaluable in expanding our comprehension of uncommon clinical presentations in animals. Animal models for human diseases such as Duchenne muscular dystrophy 15 and neuronal ceroid lipofuscinosis 16 have been discovered using WGS, which could aid in preclinical testing of therapeutics to ultimately benefit both humans and veterinary species with these conditions. Genomic evaluation of affected familial counterparts could provide the genetic etiology and heritability patterns of diseases, thus allowing the ability to screen future litters for such conditions as well as whether they might be amenable to novel therapeutic techniques such as gene therapy. A major limitation of this case is a lack of ciliary analysis to appropriately determine whether dysfunction or aplasia was present. Anecdotally, no other members of this dog's family exhibited similar clinical features, suggesting a de novo rather than a congenital mutation; however, further familial genetic or proteomic studies of dogs with this mutation would be required to determine the heritability of this mutation as well as its functional impact. The relative scarcity of reports of NAT10 mutations in the human literature would suggest de novo mutation, although this could reflect the general paucity of WGS of human patients with similar presenting clinical signs. Another limitation is the lack of NAT10 expression analysis in this dog's tissues to determine the presence of aberrant NAT10 protein. Limitations of WGS such as cost and equipment availability currently preclude extensive testing of animals, but by better streamlining this process, it could 1 day be possible to utilize WGS more readily to improve our understanding of both common and uncommon disease presentations.

| SUMMARY
This study has identified a novel mutation of the NAT10 gene in a young Schnauzer dog with chronic respiratory tract infections, gastrointestinal tract abnormalities, and chronic leukocytosis. In silico analysis of this mutation predicted it to be deleterious, and despite aggressive supportive care, the dog was euthanized due to a poor quality of life. Consideration of NAT10 mutation should be given to similar case presentations, and whole genome sequencing could aid in confirmation of this diagnosis. Further identification of this mutation in dogs might help to elucidate its heritability, any beneficial therapies for these dogs, and to possibly identify a comparable condition in humans that could be used as a reference for treatment.

ACKNOWLEDGMENT
No funding was received for this study.

CONFLICT OF INTEREST DECLARATION
Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
Authors declare no IACUC or other approval was needed.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.