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A deletion of IDUA exon 10 in a family of Golden Retriever dogs with an attenuated form of mucopolysaccharidosis type I

Abstract Background Mucopolysaccharidosis type I (MPS‐I) is a lysosomal storage disorder caused by a deficiency of the enzyme α‐l‐iduronidase, leading to accumulation of undegraded dermatan and heparan sulfates in the cells and secondary multiorgan dysfunction. In humans, depending upon the nature of the underlying mutation(s) in the IDUA gene, the condition presents with a spectrum of clinical severity. Objectives To characterize the clinical and biochemical phenotypes, and the genotype of a family of Golden Retriever dogs. Animals Two affected siblings and 11 related dogs. Methods Family study. Urine metabolic screening and leucocyte lysosomal enzyme activity assays were performed for biochemical characterization. Whole genome sequencing was used to identify the causal mutation. Results The clinical signs shown by the proband resemble the human attenuated form of the disease, with a dysmorphic appearance, musculoskeletal, ocular and cardiac defects, and survival to adulthood. Urinary metabolic studies identified high levels of dermatan sulfate, heparan sulfate, and heparin. Lysosomal enzyme activities demonstrated deficiency in α‐l‐iduronidase activity in leucocytes. Genome sequencing revealed a novel homozygous deletion of 287 bp resulting in full deletion of exon 10 of the IDUA gene (NC_006585.3(NM_001313883.1):c.1400‐76_1521+89del). Treatment with pentosan polyphosphate improved the clinical signs until euthanasia at 4.5 years. Conclusion and Clinical Importance Analysis of the genotype/phenotype correlation in this dog family suggests that dogs with MPS‐I could have a less severe phenotype than humans, even in the presence of severe mutations. Treatment with pentosan polyphosphate should be considered in dogs with MPS‐I.

Various naturally occurring forms of MPS have been described in veterinary medicine including in the dog (MPS I, II, IIIA, IIIB, VI, VII), [3][4][5][6][7][8][9] cat (MPS I, VI, VII), [10][11][12] farm animals (MPS IIIB and IIID), 13,14 and emus (MPS IIIB) 15 ; research colonies have been established for some of MPS subtypes and are currently used as animal models of their human counterparts. 16,17 We describe here the clinicopathologic findings of naturally occurring MPS-I in a Golden Retriever family, which we found to be caused by a full deletion of exon 10 of the gene coding for alpha-Liduronidase (IDUA).

| Animal cases and ethical statement
The proband was presented to the University of Glasgow Small Animal Hospital for chronic diarrhea, weight loss and dysmorphism. Diagnostic investigations were performed as part of clinical procedures. A female littermate of the proband had previously been presented to the same institution for orthopedic signs and dysmorphism, for which no diagnosis was achieved, and she was soon thereafter euthanized at her owner request and no necropsy was performed. The breeder of the affected dogs and the owners of related dogs were contacted and agreed to provide DNA samples (cheek swabs) and pedigree information from their animals ( Figure 1). The study was approved by the local ethical committee (University of Glasgow, School of Veterinary Medicine-Form ref 55a/15).

| Clinical investigations
Routine investigations included CBC, routine blood biochemistry, urinalysis, fecal culture, and diagnostic imaging tests (abdominal and cardiac ultrasound examinations and thoracic radiographs).

| Urinary metabolic studies and leucocyte lysosomal enzyme activities
Glycosaminoglycans were measured in the urine of the proband and a normal control dog for comparison. Urinary glycosaminoglycan concentration was determined using a colorimetric protocol as previously described. 18 Briefly glycosaminoglycans (GAGs) form complex molecules in the presence of the dye 1,9-dimethylmethylene blue (Sigma, UK) in acid solution. This produces a color change from blue to pink, which can be quantified at 520 nm against a standard of known concentration. Extracted GAGs were analyzed by 2-dimensional electrophoresis on cellulose acetate membranes and then visualized by staining with 0.05% alcian blue stain (Sigma, UK) to differentiate sulfated glycosaminoglycan patterns as previously described. 19 Oligosaccharide and sialic acid analysis of urine was performed by thin layer chromatography (TLC) using a previously published protocol. 20 Urine oligosaccharides were separated on thin layer slica gel plates using a mobile phase of butanol/acetic acid/water (ratio 2 : 1 : 1) and then visualized by 0.2% orcinol and heat 100 C. Urine sialic acids were separated on thin layer slica gel plates using an initial mobile phase of butanol/acetic acid/water (ratio 2 : 1 : 1) followed by propan-1-ol/nitromethane/water (ratio 5 : 4 : 3) and then visualized by 0.2% recorcinol and heat 100 C.
The activities of lysosomal enzymes were measured in leucocytes from the proband and a normal control dog using commercially available 4-methylumbelliferyl substrates (Glycosynth, UK). The activity of α-L-iduronidase was measured in leucocytes using 2 mM 4-methylumbelliferyl-α-L-iduronide (Glycosynth, UK) in 0.4 M formate buffer pH 3.5 containing 0.9% sodium chloride, using the previously published protocol. 21 Arylsulfatase B activity was measured in leucocytes with 4-methylumbelliferyl-sulfate (Glycosynth, UK) 5 mg/mL in 0.1 M acetate buffer pH 5.4 according to the previously published protocol. 22 The activity of β-glucuronidase was measured in leucocytes using the substrate 5 mM 4-methylumbelliferyl-β-Dglucuronide (Glycosynth, UK) in 0.1 M acetate buffer pH 4.0. Briefly sample lysate 10 μL was incubated with 20 μL substrate for 15 minutes at 37 C. The reaction was terminated by the addition of 0.2 M carbonate buffer pH 9.5.
For each assay all incubations were performed at 37 C, and in each case the liberated 4-methylumbelliferone (4-MU) was measured using a LS55 luminescence spectrophotometer set at emission wavelength 450 nm and excitation wavelength 360 nm (PerkinElmer), against a 4-methylumbelliferone standard of known concentration (Sigma). The total protein was measured in cell lysate supernatants using the standard Lowry total protein assay and all enzyme activities were expressed as nmol 4-MU generated/mg total protein/hour.

| Postmortem analysis
Following euthanasia, a full postmortem examination was performed.
Representative samples from the lungs, spleen, kidney, urinary bladder, liver, heart and pericardium, adrenal gland, pancreas, thyroid, tongue, gastrointestinal tract, prostate, testis, skin, sciatic and vagus nerves, brain (multiple sections) and eye were fixed in 10% buffered formalin and snap frozen in isopentane chilled in liquid nitrogen. Slices of formalin-fixed samples were then routinely processed and embedded in paraffin before sectioning and staining with hematoxylin and eosin (H&E). Additionally, formalin-fixed tissue was also cut as frozen sections and stained with Oil Red O, Luxol fast blue (LFB) and periodic acid-Schiff stain (PAS).

| Molecular analysis
DNA from the affected animal was extracted from blood using a DNeasy Blood and Tissue kit (Qiagen, Crawley, UK). For the family relatives, DNA was extracted from cheek swabs using Gentra Puregene Buccal Cell Kit (Qiagen, Crawley, UK). Following PCR amplification (see Table S1), purification of the PCR products was performed Sequencing traces were analyzed with Sequencher software v.4.2 (Gene Codes). However, due to the very high GC content of the region, some exons could not be amplified by PCR or Sanger sequencing (see Table S1) and whole-genome sequencing was then carried out by the Edinburgh Genomics laboratories, University of Edinburgh, using Illumina HiSeq X. Sequence alignment was performed against the dog genome reference CanFam3.1 using bwa 23  In addition, the genomes of 548 dogs from 107 dog breeds were screened for this specific variant 24 (see Table S2 for further information on the dog breeds).

| Clinical history and examination
A 3 year and 6 month-old male Golden Retriever was presented to the Small Animal Hospital at the University of Glasgow with an F I G U R E 1 Pedigree of the affected dogs. Squares represent males and circle females. Affected individuals are depicted with the plain black symbols with the arrow highlighting the proband and black circle representing the suspected affected littermate (not genetically tested). Unaffected animals are represented with the white symbols. The genotype of the individuals is noted below each symbol: +/+ for dogs homozygous for the wild-type allele, +/− for heterozygous dogs, −/− for dogs homozygous for the variant lacking exon 10, and ? for nontested dogs 8-month history of diarrhea and weight loss and a long-standing history of dysmorphism. On presentation, the dog was alert and responsive but was markedly underweight (body condition score 2/9; body weight 21 kg). General examination revealed a dysmorphic appearance ( Figure 2A); the proband was subjectively smaller than his littermates and had a disproportionately large head and tongue, prognathism, and a curved muzzle ( Figure 2B). Excessive amount of soft tissue was also noted on the left caudal aspect of the tongue. On cardiac auscultation, a 1/6 systolic murmur with split S2 and occasional premature beats with pulse deficit were noted. The dog was also suffering from lipid deposits in both eyes ( Figure 2C) and interdigital cysts between the fourth and fifth digits in all limbs ( Figure 2D). Abnormal findings on neurological examination consisted in bilateral positional ventral strabismus and nystagmus (of variable direction). This dog originated from a litter of 8 puppies, of which a female littermate was euthanized a few months earlier. She exhibited similar dysmorphic changes and corneal lipid deposits; however, no diagnosis was achieved at that time. In both dogs, abnormal limb conformation was first noted by the breeder from the age of 3 to 4 months, and in the euthanized bitch multiple joint laxities (carpi and stifles) were noted by a veterinary orthopedic surgeon when she was 9 months old.

| Biochemical confirmation of diagnosis
Quantification of urinary oligosaccharides and sialic acid was within normal limits.
Urinary glycosaminoglycan analysis revealed a marked increase in concentrations of dermatan and heparan sulfates ( Figure S1).
The activities of the relevant lysosomal enzymes are shown in Table 1. The marked deficiency in α-L-iduronidase activity, reduced to 1.3% of the normal control dog activity, was consistent with a diagnosis of MPS-I. Since a biochemical diagnosis of MPS-I had been established, the activity of iduronate-2-sulfatase was not measured.  Figure 4A). Bilaterally, the vagus nerves appeared thickened and firm. The prostate gland was symmetrically and mildly to moderately enlarged.

| Treatment and outcome
The histological changes were consistent with a common pattern of accumulation of myxomatous material and fibrosis. These myxomatous changes were most evident in all heart valves, the presumed vagus nerves (histologically no nervous tissue was appreciable anymore), parts of the sciatic nerve and predominantly the tunica intima, less so the tunica media of large vessels, whereas fibrosis was evident in the pulmonary pleura, the pericardium, the epicardium, the leptomeninges, the adrenal cortex and also the medulla of the peripheral lymph node examined. Numerous organs, including the spleen, kidney, urinary bladder, liver, gastrointestinal system, lymph nodes, skin, nerves, leptomeninges, cornea, and also the collagenous tissue forming growths extending from the tongue and overlying the epicardium contained foamy macrophages ( Figure S2 The interstitial Leydig cells were also vacuolated ( Figure 4D). Finally, histological changes consistent with prostatic hyperplasia were observed.  (Table S2).

| Genetic analysis
The variant was not found in any of the analyzed dog genomes.

| DISCUSSION
In this study, we describe a Golden Retriever family affected by MPS-I secondary to a full deletion of the IDUA exon 10. The proband tal, cardiac and ocular as seen in the Plott Hound model. 52 Facial changes were also striking with a curved muzzle typically seen in affected dogs 53 and a mandibular prognatism, also described in humans. 54 Moreover, while no marked behavioral change was identified by the owner or the clinician, subtle neurological signs were detected on neurological examination. Finally, the dog also reached reproductive age. In human patients with 2 null variants of IDUA such as a nonsense mutation or a large deletion, a severe phenotype is consistently observed. 43,46 This would suggest that the phenotype in the dog models is attenuated compared to what would be expected in humans suffering from nonsense mutations. A similar phenotype attenuation has been discussed in the murine knock-out 30 and spontaneous feline model, 29 which also reached adulthood. This is an interesting finding highlighting the limitations of animal models, which do not always fully reproduce the human phenotype. The reason why these animals do not suffer from a severe form remains unknown and could be due to subtle variations in the metabolic rate of GAG depending on the species. 30 Understanding these interspecies differences could be an interesting subject of research, for which the establishment of a new dog research colony from these Golden retriever dogs could prove helpful.
The main concern of the proband's owner was the recent onset of gastrointestinal signs. Episodic diarrhea is also seen in many children with Hurler's disease. Its etiology remains unknown although abnormal intestinal flora has been suggested. 55 The proband also F I G U R E 6 Integrated Genomics Viewer (IGV) display of the 3 0 end of the IDUA gene sequencing data of the affected dog. The lower panel of the display shows the normal structure of the gene with the location of exons 3 to 14. In the affected dog, there is a deletion of the entire exon 10 suffered from renal agenesis. To the best of our knowledge, this has never been described in people suffering from MPS-I and in this case very likely represents an incidental and unrelated finding. However, we cannot fully exclude that this could be a previously unseen sign.
Interestingly, histopathology revealed testicular changes, which is poorly documented in people with MPS although testicular histopathological changes were observed in a 19 year old man suffering from MPS-II. 56 Recently, male reproductive function was assessed in a murine model of MPS-I. In these mice, tubular degeneration and disorganization of the seminiferous epithelium with tubules containing very few germ cells were observed histologically, similar to the findings observed in this dog. Consistent with the histological findings, these mice exhibited a marked reduction in the daily sperm production but the plasma testosterone concentrations remained normal. 57 The fact that testosterone concentrations remain unchanged would explain the benign prostatic hyperplasia seen in the proband. Larger animal models such as cats and dogs have a longer life expectancy, making it more appropriate to test therapeutics including their potential complications in the long term. 16,68 Their size and neuroanatomy (gyrencephalic vs lissencephalic brain) is also closer to that of a young child, allowing better assessment of more advanced treatment such as stereotaxic injections. 53,68,69 In the present case, the prohibitive cost of ERT precluded its use, all the more as weekly injections are typically required. 28 Experimental treatment with PPS has recently been shown to have a beneficial effect in the rat model of MPS-VI and dog model of MPS-I, resulting in a marked reduction of the associatedinflammation but also a reduction in the accumulation of GAG in the tissues and urine. [70][71][72] Although improvement of quality of life was reported by the owner of the current dog after the treatment was initiated, this remains subjective.
In conclusion, we have described a dog with MPS-I, which resembles the attenuated form of the human disease. In this dog, the disease is secondary to a full deletion of the exon 10 of the IDUA gene, leading to a deficiency in α-L-iduronidase activity. Contrary to what would be expected in human patients with such a severe mutation, the phenotype of this dog remained attenuated.