Identification of Novel Mutations and Molecular Pathways for Canine Neurodegeneration and Chondrodysplasia

Inherited diseases occur across different species. This thesis work has provided insights into the molecular genetic background of three autosomal recessive diseases that affect specific dog breeds. The studied phenotypes comprised two neurodegenerative diseases and a type of skeletal dysplasia. Genome-wide methods, such as SNP chip genotyping, were used to identify disease-associated gene variants. In all three disease phenotypes, the likely causative variant was found in a gene that had not been previously associated with a monogenic disorder. The Norwegian Elkhound and Karelian Bear Dog breeds are affected with inherited chondrodysplasia that causes short-stature dwarfism of varying severity. A genome-wide association study in Norwegian Elkhounds revealed a disease-associated locus on canine chromosome 17 and a nonsense mutation in the ITGA10 gene. The identified mutation was homozygous in all affected dogs from both breeds, and may have been introduced to Karelian Bear Dogs from Norwegian Elkhounds. The ITGA10 gene encodes an α10-integrin protein that assembles into a collagen-binding α10β1 integrin. The α10β1 integrin is a cell surface receptor, found in growth plate chondrocytes, where it mediates the cell s attachments with the surrounding matrix. Due to the mutation, a full-length α10-protein is not produced, disturbing the growth of long bones. Early-onset cerebellar degeneration occurs in the Finnish Hound dog breed. Neurological examination of affected dogs revealed quickly progressing cerebellar ataxia and failure to thrive. Genome-wide association analyses mapped the disease to a 1.5-Mb locus on canine chromosome 8. Sequencing of the SEL1L gene from the locus identified a homozygous missense mutation in all affected dogs. The mutation causes a serine to proline amino acid change within a highly conserved functional domain of the encoded SEL1L protein. The SEL1L protein is found in the endoplasmic reticulum, where it functions within a protein quality control and degradation pathway. Cerebellar tissue samples from affected dogs showed signs of endoplasmic reticulum stress, which may be the cause of premature cell death. A novel neurological disease, characterized by juvenile to adult onset cerebellar ataxia, was recognized in the Lagotto Romagnolo breed. Through linkage analysis, homozygosity mapping and whole-genome sequencing, the disease was associated with a homozygous missense change in the autophagy-related ATG4D gene. Pathological examination of affected dogs revealed progressive cerebellar degeneration, and intracellular vacuolar changes both in neuronal and extraneuronal tissues. The ATG4D gene encodes a cysteine protease, which is thought to function in the macroautophagy pathway. The autophagy process degrades and recycles damaged or obsolete cellular materials via membrane-enclosed autophagosomes. In line with this, the neuronal tissues of affected dogs showed signs of altered autophagic flow. Overall, this study has revealed three new disease-linked genes in dogs, which may be associated to similar disorders in other species. On the basis of the results, DNA-tests have been developed for veterinary diagnostic and breeding purposes. Importantly, by shedding light into disease-causing pathways, the results of this study could prove beneficial not just for canine health but for human medicine as well.Perinnöllisiä sairauksia tavataan ihmisten lisäksi myös muissa lajeissa, kuten kotieläimissä. Tässä väitöskirjatyössä tutkittiin kolmea koirissa esiintyvää peittyvästi periytyvää sairautta. Tutkimuksen kohteena oli lyhytkasvuisuutta aiheuttava luustosairaus sekä kaksi erityyppistä hermostorappeumasairautta. Tutkimuksessa käytettiin hyväksi koko perimän laajuisia tutkimusmenetelmiä, joiden avulla paikannettiin sairauksien taustalla olevat geenivirheet. Harmaissa norjanhirvikoirissa sekä karjalankarhukoirissa esiintyy kondrodysplasiaa, eli rustonsisäisen luutumisen häiriötä, joka ilmenee lyhytkasvuisuutena. Sairaiden koirien raajat ovat huomattavan lyhyet ja näillä esiintyy myös muita vakavuudeltaan vaihtelevia luustomuutoksia. Kummankin rodun lyhytkasvuisilta koirilta löytyi homotsygoottinen geenivirhe ITGA10-geenistä. Tutkimustulosten perusteella geenivirhe on saattanut siirtyä karjalankarhukoiriin harmaista norjanhirvikoirista. ITGA10-geeni koodaa α10-intergriini proteiinia, joka on osa kasvulevyjen rustosoluissa toimivaa, kollageeneja sitovaa α10β1-integriini reseptoria. Geenivirhe estää α10-proteiinin normaalin tuoton, minkä seurauksena kasvulevyn toiminta sekä luuston kasvu häiriintyy. Suomenajokoirilla tavataan pentuiän pikkuaivorappeumaa, jota aiheuttava geenivirhe löytyi SEL1L-geenistä. Sairaat ajokoiranpennut kärsivät nopeasti etenevästä pikkuaivoataksiasta, eli liikkeiden säätelyn vaikeudesta. Pennuilta tunnistettiin SEL1L-geenistä homotsygoottinen yhden emäksen muutos, joka johtaa yhden aminohapon vaihtumiseen toiseksi SEL1L-proteiinin toiminnallisesti tärkeässä rakenteessa. SEL1L-proteiini toimii solulimakalvostossa proteiinien laaduntarkkailussa. Tunnistetun geenivirheen oletetaan aiheuttavan häiriöitä solulimakalvossa, mikä puolestaan vaikuttaa pikkuaivojen hermosolujen solunsisäinen tasapainoon ja johtaa ennenaikaiseen solukatoon. Lagotto romagnolo -rodusta tunnistettiin uuden tyypin neurologinen sairaus, jonka pääasiallinen oire on etenevä, nuoruus- tai aikuisiällä alkava pikkuaivoataksia. Patologiset tutkimukset paljastivat solutason muutoksia paitsi keskushermostosta, mutta myös muualta elimistöstä. Pikkuaivoissa nähtiin etenevää hermosolukatoa. Sairauteen liitettiin homotsygoottinen yhden emäksen muutos ATG4D-geenissä. ATG4D-geenin toiminta liittyy autofagosytoosiin, joka on solunsisäinen puhdistus- ja ravinteiden kierrätys mekanismi. Sairaiden koirien keskushermostosta löytyikin viitteitä autofagosytoosin häiriintymisestä. Kyseisiä geenejä ei ole aiemmin liitetty vastaaviin sairauksiin muissa lajeissa, mikä on antanut uutta tietoa sairauksiin liittyvistä molekyylireiteistä. Tästä tiedosta saattaa tulevaisuudessa olla hyötyä myös ihmislääketieteessä. Tutkimustulokset ovat lisäksi mahdollistaneet geenivirheitä kantavien koirien tunnistamisen DNA-testeillä. Testauksen avulla voidaan estää sairaiden koirien syntyminen sekä saada tukea eläinlääketieteelliseen diagnostiikkaan

Canine Chondrodysplasia Caused by a Truncating Mutation in Collagen-Binding Integrin Alpha Subunit 10

Peer reviewe

An across-breed validation study of 46 genetic markers in canine hip dysplasia

BackgroundCanine hip dysplasia (CHD) is a common disease, with a complex genetic background. Dogs with severe CHD sometimes also suffer from osteoarthritis (OA), an inflammatory, often painful and incurable condition. Previous studies have reported breed-specific genetic loci associated with different hip dysplasia and OA phenotypes. However, the independent replication of the known associations within or across breeds has been difficult due to variable phenotype measures, inadequate sample sizes and the existence of population specific variants.ResultsWe execute a validation study of 46 genetic markers in a cohort of nearly 1600 dogs from ten different breeds. We categorize the dogs into cases and controls according to the hip scoring system defined by the Federation Cynologique Internationale (FCI). We validate 21 different loci associated on fourteen chromosomes. Twenty of these associated with CHD in specific breeds, whereas one locus is unique to the across-breed study. We show that genes involved in the neddylation pathway are enriched among the genes in the validated loci. Neddylation contributes to many cellular functions including inflammation.ConclusionsOur study successfully replicates many loci and highlights the complex genetic architecture of CHD. Further characterisation of the associated loci could reveal CHD-relevant genes and pathways for improved understanding of the disease pathogenesis.Peer reviewe

Recessive missense LAMP3 variant associated with defect in lamellar body biogenesis and fatal neonatal interstitial lung disease in dogs

Neonatal interstitial lung diseases due to abnormal surfactant biogenesis are rare in humans and have never been reported as a spontaneous disorder in animals. We describe here a novel lung disorder in Airedale Terrier (AT) dogs with clinical symptoms and pathology similar to the most severe neonatal forms of human surfactant deficiency. Lethal hypoxic respiratory distress and failure occurred within the first days or weeks of life in the affected puppies. Transmission electron microscopy of the affected lungs revealed maturation arrest in the formation of lamellar bodies (LBs) in the alveolar epithelial type II (AECII) cells. The secretory organelles were small and contained fewer lamellae, often in combination with small vesicles surrounded by an occasionally disrupted common limiting membrane. A combined approach of genome-wide association study and whole exome sequencing identified a recessive variant, c.1159G>A, p.(E387K), in LAMP3, a limiting membrane protein of the cytoplasmic surfactant organelles in AECII cells. The substitution resides in the LAMP domain adjacent to a conserved disulfide bond. In summary, this study describes a novel interstitial lung disease in dogs, identifies a new candidate gene for human surfactant dysfunction and brings important insights into the essential role of LAMP3 in the process of the LB formation. Author summary We have characterized a lethal lung disease in neonatal Airedale Terrier dogs. The pathological features of the disease resemble those of the surfactant dysfunction in newborn babies. Surfactant is essential for lung function and we observed a maturation defect in the surfactant producing organelles of the lung epithelial type II cells. Genetic analyses revealed a recessive variant in the lysosome associated membrane LAMP3 gene. LAMP3 is a structural, limiting membrane protein of the surfactant organelles. This study provides an excellent candidate gene for human surfactant disorders as well as new insights into LAMP3 biology and pathophysiology while the affected breed will benefit from genetic testing to eradicate this severe disease.Peer reviewe

A homozygous missense variant in the alkaline phosphatase gene ALPL is associated with a severe form of canine hypophosphatasia

Inherited skeletal disorders affect both humans and animals. In the current study, we have performed series of clinical, pathological and genetic examinations to characterize a previously unreported skeletal disease in the Karelian Bear Dog (KBD) breed. The disease was recognized in seven KBD puppies with a variable presentation of skeletal hypomineralization, growth retardation, seizures and movement difficulties. Exome sequencing of one affected dog revealed a homozygous missense variant (c. 1301T > G; p. V434G) in the tissue non-specific alkaline phosphatase gene, ALPL. The identified recessive variant showed full segregation with the disease in a cohort of 509 KBDs with a carrier frequency of 0.17 and was absent from 303 dogs from control breeds. In humans, recessive and dominant ALPL mutations cause hypophosphatasia (HPP), a metabolic bone disease with highly heterogeneous clinical manifestations, ranging from lethal perinatal hypomineralization to a relatively mild dental disease. Our study reports the first naturally occurring HPP in animals, resembling the human infantile form. The canine HPP model may serve as a preclinical model while a genetic test will assist in breeding programs.Peer reviewe

Altered Basal Autophagy Affects Extracellular Vesicle Release in Cells of Lagotto Romagnolo Dogs With a Variant ATG4D

Lagotto Romagnolo breed dogs develop a progressive neurological disease with intracellular vacuolar storage when homozygous for a variant in the autophagy-related gene 4D (ATG4D). A lysosomal enzyme deficiency has not been proven in this disease, despite its overlapping morphology with lysosomal storage diseases. Instead, basal autophagy was altered in fibroblasts from affected dogs. The aim of this study was to clarify the origin of the limiting membrane of the accumulating vacuoles and determine whether altered basal autophagy affects the extracellular release of vesicles in cells from diseased dogs. When assessed by immunoelectron microscopy, the membrane of the cytoplasmic vacuoles in affected tissues contained ATG4D, markers for autolysosomes (microtubule-associated protein 1A/B light chain 3 and lysosome-associated membrane protein 2) and for recycling endosomes (transferrin receptor 2), indicating that the vacuoles are hybrid organelles between endocytic and autophagic pathways. Ultracentrifugation, nanoparticle tracking analysis, and mass spectrometry were used to analyze the vesicles released from cultured fibroblasts of affected and control dogs. The amount of extracellular vesicles (EVs) released from affected fibroblasts was significantly increased during basal conditions in comparison to controls. This difference disappeared during starvation. The basal EV proteome of affected cells was enriched with cytosolic, endoplasmic reticulum, and mitochondrial proteins. Heat shock proteins and chaperones, some of which are known substrates of basal autophagy, were identified among the proteins unique to EVs of affected cells. An increased release of extracellular vesicles may serve as a compensatory mechanism in disposal of intracellular proteins during dysfunctional basal autophagy in this spontaneous disease.Peer reviewe

A putative silencer variant in a spontaneous canine model of retinitis pigmentosa

Author summary Retinitis pigmentosa (RP) is a blinding eye disease that affects nearly two million people worldwide. Several genes and variants have been associated with the disease, but still 30-80% of the patients lack genetic diagnosis. There is currently no standard treatment for RP, and much is expected from gene therapy. A similar disease, called progressive retinal atrophy (PRA), affects many dog breeds. We performed clinical, genetic and functional analyses to find the genetic cause for PRA in Miniature Schnauzers. We discovered two forms of PRA in the breed, named type 1 and 2, and show that they are genetically distinct as they map to different chromosomes, 15 and X, respectively. Further genetic, bioinformatic and functional analyses discovered a fully penetrant recessive variant in a putative silencer region for type 1 PRA. Silencer regions are important for gene regulation and we found that two of its predicted target genes, EDN2 and COL9A2, were overexpressed in the retina of the affected dog. Defects in both EDN2 and COL9A2 have been associated with retinal degeneration. This study provides new insights to retinal biology while the genetic test guides better breeding choices. Retinitis pigmentosa (RP) is the leading cause of blindness with nearly two million people affected worldwide. Many genes have been implicated in RP, yet in 30-80% of the RP patients the genetic cause remains unknown. A similar phenotype, progressive retinal atrophy (PRA), affects many dog breeds including the Miniature Schnauzer. We performed clinical, genetic and functional experiments to identify the genetic cause of PRA in the breed. The age of onset and pattern of disease progression suggested that at least two forms of PRA, types 1 and 2 respectively, affect the breed, which was confirmed by genome-wide association study that implicated two distinct genomic loci in chromosomes 15 and X, respectively. Whole-genome sequencing revealed a fully segregating recessive regulatory variant in type 1 PRA. The associated variant has a very recent origin based on haplotype analysis and lies within a regulatory site with the predicted binding site of HAND1::TCF3 transcription factor complex. Luciferase assays suggested that mutated regulatory sequence increases expression. Case-control retinal expression comparison of six best HAND1::TCF3 target genes were analyzed with quantitative reverse-transcriptase PCR assay and indicated overexpression of EDN2 and COL9A2 in the affected retina. Defects in both EDN2 and COL9A2 have been previously associated with retinal degeneration. In summary, our study describes two genetically different forms of PRA and identifies a fully penetrant variant in type 1 form with a possible regulatory effect. This would be among the first reports of a regulatory variant in retinal degeneration in any species, and establishes a new spontaneous dog model to improve our understanding of retinal biology and gene regulation while the affected breed will benefit from a reliable genetic testing.Peer reviewe

TSEN54 missense variant in Standard Schnauzers with leukodystrophy

We report a hereditary leukodystrophy in Standard Schnauzer puppies. Clinical signs occurred shortly after birth or started at an age of under 4 weeks and included apathy, dysphoric vocalization, hypermetric ataxia, intension tremor, head tilt, circling, proprioceptive deficits, seizures and ventral strabismus consistent with a diffuse intracranial lesion. Magnetic resonance imaging revealed a diffuse white matter disease without mass effect. Macroscopically, the cerebral white matter showed a gelatinous texture in the centrum semiovale. A mild hydrocephalus internus was noted. Histopathologically, a severe multifocal reduction of myelin formation and moderate diffuse edema without inflammation was detected leading to the diagnosis of leukodystrophy. Combined linkage analysis and homozygosity mapping in two related families delineated critical intervals of approximately 29 Mb. The comparison of whole genome sequence data of one affected Standard Schnauzer to 221 control genomes revealed a single private homozygous protein changing variant in the critical intervals, TSEN54:c.371G>A or p.(Gly124Asp). TSEN54 encodes the tRNA splicing endonuclease subunit 54. In humans, several variants in TSEN54 were reported to cause different types of pontocerebellar hypoplasia. The genotypes at the c.371G>A variant were perfectly associated with the leukodystrophy phenotype in 12 affected Standard Schnauzers and almost 1000 control dogs from different breeds. These results suggest that TSEN54:c.371G>A causes the leukodystrophy. The identification of a candidate causative variant enables genetic testing so that the unintentional breeding of affected Standard Schnauzers can be avoided in the future. Our findings extend the known genotype-phenotype correlation for TSEN54 variants.Peer reviewe

Characterisation of canine KCNIP4: A novel gene for cerebellar ataxia identified by whole-genome sequencing two affected Norwegian Buhund dogs

Author summary Hereditary ataxias, which are a group of disorders characterised by incoordination of movement, are typically incurable and there are often no disease-modifying treatments available. Canine hereditary ataxias are a notable group of movement disorders in dogs, and represent well characterised naturally occurring disease models of ataxia that can help improve our understanding of the underlying biology of the disorder in both dogs and humans. We used the whole genome sequences of two affected siblings to investigate the genetic cause of a slowly progressive form of hereditary ataxia in the Norwegian Buhund dog breed, and identified a single base change within the KCNIP4 gene. We have characterised the expression of KCNIP4 in the dog, and investigated the effect of the identified mutation. This gene has not previously been implicated in inherited ataxia in any species, and our findings suggest that this and related genes represent potential candidates for ataxia in future studies in other species. Our findings will allow dog breeders to avoid producing affected dogs, reduce the disease allele frequency, and eventually eliminate the disease from the breed, through the use of a DNA test. A form of hereditary cerebellar ataxia has recently been described in the Norwegian Buhund dog breed. This study aimed to identify the genetic cause of the disease. Whole-genome sequencing of two Norwegian Buhund siblings diagnosed with progressive cerebellar ataxia was carried out, and sequences compared with 405 whole genome sequences of dogs of other breeds to filter benign common variants. Nine variants predicted to be deleterious segregated among the genomes in concordance with an autosomal recessive mode of inheritance, only one of which segregated within the breed when genotyped in additional Norwegian Buhunds. In total this variant was assessed in 802 whole genome sequences, and genotyped in an additional 505 unaffected dogs (including 146 Buhunds), and only four affected Norwegian Buhunds were homozygous for the variant. The variant identified, a T to C single nucleotide polymorphism (SNP) (NC_006585.3:g.88890674T>C), is predicted to cause a tryptophan to arginine substitution in a highly conserved region of the potassium voltage-gated channel interacting protein KCNIP4. This gene has not been implicated previously in hereditary ataxia in any species. Evaluation of KCNIP4 protein expression through western blot and immunohistochemical analysis using cerebellum tissue of affected and control dogs demonstrated that the mutation causes a dramatic reduction of KCNIP4 protein expression. The expression of alternative KCNIP4 transcripts within the canine cerebellum, and regional differences in KCNIP4 protein expression, were characterised through RT-PCR and immunohistochemistry respectively. The voltage-gated potassium channel protein KCND3 has previously been implicated in spinocerebellar ataxia, and our findings suggest that the Kv4 channel complex KCNIP accessory subunits also have an essential role in voltage-gated potassium channel function in the cerebellum and should be investigated as potential candidate genes for cerebellar ataxia in future studies in other species.Peer reviewe

An across-breed validation study of 46 genetic markers in canine hip dysplasia

Abstract Background Canine hip dysplasia (CHD) is a common disease, with a complex genetic background. Dogs with severe CHD sometimes also suffer from osteoarthritis (OA), an inflammatory, often painful and incurable condition. Previous studies have reported breed-specific genetic loci associated with different hip dysplasia and OA phenotypes. However, the independent replication of the known associations within or across breeds has been difficult due to variable phenotype measures, inadequate sample sizes and the existence of population specific variants. Results We execute a validation study of 46 genetic markers in a cohort of nearly 1600 dogs from ten different breeds. We categorize the dogs into cases and controls according to the hip scoring system defined by the Fédération Cynologique Internationale (FCI). We validate 21 different loci associated on fourteen chromosomes. Twenty of these associated with CHD in specific breeds, whereas one locus is unique to the across-breed study. We show that genes involved in the neddylation pathway are enriched among the genes in the validated loci. Neddylation contributes to many cellular functions including inflammation. Conclusions Our study successfully replicates many loci and highlights the complex genetic architecture of CHD. Further characterisation of the associated loci could reveal CHD-relevant genes and pathways for improved understanding of the disease pathogenesis