SCN9A variant in a family of mixed breed dogs with congenital insensitivity to pain

Abstract Background Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSANs) are a rare group of genetic disorders causing inability to feel pain. Three different associated variants have been identified in dogs: 1 in Border Collies, 1 in mixed breed dogs, and 1 in Spaniels and Pointers. Objectives To clinically and genetically characterize CIP in a family of mixed breed dogs. Animals Two mixed breed dogs from the same litter were independently presented: 1 for evaluation of painless fractures, and the other for chronic thermal skin injuries. Methods Physical, neurological, and histopathological evaluations were performed. Whole genome sequencing of 1 affected dog was used to identify homozygous protein‐changing variants that were not present in 926 control genomes from diverse dog breeds. Results Physical and neurological examinations showed the absence of superficial and deep pain perception in the entire body. Histopathological evaluations of the brain, spinal cord and sensory ganglia were normal. Whole genome sequencing identified a homozygous missense variant in SCN9A, XM_038584713.1:c.2761C>T or XP_038440641.1:(p.Arg921Cys). Both affected dogs were homozygous for the mutant allele, which was not detected in 926 dogs of different breeds. Conclusions and Clinical Importance We confirmed the diagnosis of CIP in a family of mixed breed dogs and identified a likely pathogenic variant in the SCN9A gene. The clinical signs observed in these dogs mimic those reported in humans with pathogenic SCN9A variants causing CIP. This report is the first of a spontaneous pathogenic SCN9A variant in domestic animals.


| INTRODUCTION
Pain is a sensory modality used to detect potential and real tissue damage, providing a survival advantage. 1,2 Genetic pain loss disorders are classified as congenital insensitivity to pain (CIP) or hereditary sensory and autonomic neuropathy (HSAN). 2,3 Congenital insensitivity to pain usually is defined by its congenital onset, whereas HSAN tends to develop gradually over time, but occasionally the difference is not clearly specified, and the terms can overlap. 2 In all cases of CIP or HSAN, the consistent feature is decreased pain perception and resulting injuries. 2,3 In humans, seven forms of CIP and eight forms of HSAN have been described based on phenotype, and genetic variants in at least 26 genes have been reported. 2,3 Congenital insensitivity to pain and HSANs have been described previously in some dog breeds including French Spaniel, 4,5 English Springer Spaniel, 4,5 Pointer, 4,6-8 Border Collie, [9][10][11][12] Border Collie cross, 13 Miniature Pincher, 14 Long-haired Dachshund, 15,16 Jack Russell Terrier, 17 Fox Terrier, 18 and a family of mixed breed dogs, 19 but only three causal genetic variants have been identified to date. The first is an inversion disrupting RETREG1 (reticulophagy regulator 1) in Border Collies and Border Collie crosses with HSAN (OMIA 002032-9615). 9,13 The second is a missense variant in the same gene in a family of mixed breed dogs with HSAN (OMIA 002032-9615). 19 The third variant is a regulatory single base substitution in a lincRNA upstream of the GDNF (glial cell-derived neurotrophic factor) gene encoding glial cell-derived neurotrophic factor in Pointers, English Springer Spaniels and French Spaniels with acral mutilation syndrome (OMIA 001514-9615). 4 We investigated 2 mixed breed puppies from the same litter. One had tibial and fibular fractures and was weight-bearing with the leg bending and no signs of pain. The other had chronic skin injuries caused by burns from sleeping in contact to the heating radiator. Here we describe the clinical presentation, histopathological features, outcome, and genetic investigations of these cases, in which we found a homozygous missense variant in SCN9A, XM_038584713.1: c.2761C>T or XP_038440641.1:(p.Arg921Cys). The SCN9A (sodium voltage-gated channel alpha subunit 9) gene encodes the alpha subunit of the NaV1.7 voltage-gated sodium channel, which is preferentially expressed in sensory neurons and plays a critical role in the generation and conduction of action potentials. 2 Loss-of-function mutations in this gene have been associated with complete insensitivity to pain in humans. 2

| Animals
Two related female mixed breed dogs were evaluated separately, the first 1 at 2 months of age (Case 1) and the second at 8 months of age (Case 2). They were from the same litter of reportedly healthy parents and some of the littermates also were reported to be healthy. Residual blood samples were retained from Case 2, and buccal swabs were collected from Case 1 for genetic investigations.
Samples from the dam, sire or other littermates could not be obtained.

| Necropsy examination
Owner consent was given for euthanasia and complete necropsy in Case 2. In addition to routine samples taken during necropsy (including brain and cervical, thoracic and lumbar spinal cord), representative samples from dorsal root ganglia (cervical, thoracic, and lumbar) were collected and fixed in 10% buffered formalin. Slices of formalin-fixed samples were embedded in paraffin before staining with hematoxylin and eosin.

| Variant calling
Variant calling was performed using GATK HaplotypeCaller 21 in gVCF mode as described. 20 To predict the functional effects of the called variants, SnpEff software 22 together with NCBI annotation release 106 for the UU_Cfam_GSD_1.0 genome reference assembly was used. For variant filtering, we used 926 genetically diverse control dog genomes of different breeds (Table S1).

| Gene analysis
We used the UU_Cfam_GSD_1.0 dog reference genome assembly and NCBI annotation release 106. Numbering within the canine SCN9A gene corresponds to the NCBI RefSeq accession numbers XM_038584713.1 (mRNA) and XP_038440641.1 (protein).

| Histopathology
No macroscopic or microscopic abnormalities were detected in any of the tissues examined, except for the skin lesions previously reported.

| Sequencing, genotyping and protein expression
Because the clinical and neurological findings of these cases resembled CIP previously described in humans, and the parents were F I G U R E 1 Cranio-caudal (A) and lateral (B) radiograph of the right pelvic limb of case 1 showing the tibial and fibular fractures and soft tissue swelling reported to be clinically unaffected, we hypothesized that the phenotype in the affected dogs was caused by monogenic autosomal recessive mode of inheritance. We sequenced the genome of Case 2 and searched for private homozygous variants that were not present in the genome sequences of 926 control dogs of diverse breeds (Table 1 and Tables S1 and S2). The arginine-to-cysteine substitution was predicted to be deleterious by all used prediction algorithms (PredictSNP probability for pathogenicity 87%, MutPred2 score: 0.923, SNP&GO disease probability: 76%). Furthermore, MutPred2 predictions included "altered ordered interface" and "altered transmembrane protein" with probabilities of 0.28 and 0.25, and with P-values of 5.9 Â 10 À3 and 1.4 Â 10 À3 , respectively.
The other seven private protein-changing variants were not located in genes known to cause similar phenotypes in humans, mice, or domestic animals.
We confirmed the presence of the SCN9A variant in a homozygous state in Cases 1 and 2 by Sanger sequencing (Figure 3).

| DISCUSSION
We describe two littermate mixed breed dogs with the inability to react to noxious stimuli and secondary injuries, including fractures and burns. We identified a likely candidate disease-causing variant,  28 The SCN9A gene also plays a role in seizures and epilepsy with some variants linked to Dravet Syndrome (OMIM# 607208) and febrile seizures. 29 The clinical phenotype in humans with CIP caused by variants in SCN9A is characterized by anosmia and injuries associated with complete lack of pain sensation. The dogs of our study suffered from painless fractures and burns, which are hallmarks of the phenotype in humans. Anosmia is difficult to identify clinically in dogs, especially if present since birth, and we are not sure if it was present in these cases. When compared with previous reports of HSAN and CIP in dogs, the dogs of our study share more similarities with the Miniature Pinscher, 14 Pointer, 4,6-8 Spaniel, 4,5 and Fox Terrier, 18 17 Border Collie, [9][10][11][12][13] and a family of mixed breed dogs 19 was associated with other neurological deficits including proprioceptive deficits and signs of autonomic dysfunction.
An important difference in the dogs of our report is that automutilation was not a feature, but severe injuries were caused by external sources. Finally, the young age of presentation in our patients is consistent with CIP.
Several variants in SCN9A causing CIP have been identified in humans. Congenital insensitivity to pain causing loss of function variants mostly consist of nonsense, splice site, and frameshift variants, but also include some missense variants. 26 The missense variant identified in the affected dogs of our report changes a highly conserved arginine residue in 1 of the 4 intramembrane pore-forming domains of the NaV1.7 alpha subunit. 30 The change from the positively-charged arginine residue to an uncharged cysteine residue with a reactive thiol group in this region might disrupt sodium channel functionality by altering sodium ion selectivity and conductivity, as was suggested previously. 31 The XP_038440641. arose in the recent ancestry of the affected dogs described in our study or whether it arose more distantly, in which case it might be segregating in the wider canine population.

ACKNOWLEDGMENT
No funding was received for this study. We thank the dog owners who donated samples and participated in the study. We thank the Next Generation Sequencing Platform of the University of Bern for performing the high-throughput sequencing experiments and the Interfaculty Bioinformatics Unit of the University of Bern for providing high-performance computing infrastructure. We acknowledge the Dog10K genomes project and all researchers who deposited dog or wolf whole genome sequencing data into public databases.