Koira eläinmallina silmäsairauksien geenitutkimuksessa : uusien geenien ja geenialueiden paikannus glaukoomaan ja verkkokalvonrappeumaan

The availability of the genome sequence and genomic tools combined with the unique phenotypic diversity and breed structure has increased interest in dog as a large, physiologically relevant animal model for human genetic research. Dogs suffer from hundreds of hereditary conditions of which many resemble closely disorders in humans. Investigation of the genetic background of these conditions is important to understand the disease mechanisms and pathways for therapeutic options, to identify novel candidate genes for corresponding human conditions, and to inform the breeding programs. Remarkable resources have been established in the University of Helsinki for canine genetic research, including one of the largest canine DNA banks. This study has focused on degenerative eye diseases of unknown genetic cause. We combined efforts in basic and clinical veterinary research with a unique network of collaboration with dog breeders and owners to establish clinically relevant study cohorts for two types of retinal degeneration in Papillon and Phaléne, and in Swedish Vallhund, SV breeds and two different types of glaucoma in Dandie Dinmont Terriers, DDT and Norwegian Elkhounds, NE. Genome wide approaches with the latest genomic tools, including SNP arrays and exome sequencing were then utilized to identify causative loci and genes. We report breakthroughs in all projects and highlight shared molecular etiologies in canine and human vision disorders. The cause of the retinal degeneration in Papillons and Phalénes was found in the CNGB1 gene, which is important for the rod cell function and has been linked before to human retinal degenerations. A unique type of multifocal retinal degeneration was described in SV with association to the upregulation of the MERTK gene. MERTK mutations cause also retinal degeneration in humans. DDTs were found to present a primary closed angle glaucoma (PACG) with severe pectinate ligament dysplasia (PLD). The disease was mapped to a region on canine chromosome 8, which is syntenic to human chromosome 14 with multiple glaucoma loci. However, further studies are required to identify the causative mutation. Finally, we found that the primary glaucoma in NEs is caused by a missense mutation in the ADAMTS10 gene. This gene has been associated with a Weill-Marchesani syndrome (WMS) with ocular and non-ocular abnormalities in humans. This study provides new candidate genes, and mechanisms for human eye disorders, and has identified models for potential therapeutic trials. At the same time breeding programs benefit from novel gene tests to eradicate the blinding conditions from the studied breeds.Rotukoirien voimakas jalostus on tuottanut satoja perimältään uniikkeja rotuja ja rikastanut niihin satoja perinnöllisiä sairauksia, jotka muistuttavat läheisesti vastaavia sairauksia ihmisillä. Helsingin yliopistoon on rakennettu kansainvälisesti merkittävät tutkimusresurssit geenitutkimukseen, mukaan lukien yli 50 000 näytteen geenipankki, jota on hyödynnetty tässä tutkimuksessa. Tämä tutkimus keskittyi kahteen sokeuttavaan silmäsairauteen, verkkokalvonrappeumaan sekä glaukoomaan. Verkkokalvonrappeumassa silmän näköhavainnosta vastaavat solut vaurioituvat. Glaukoomassa puolestaan verkkokalvon sähköisestä viestinnästä vastaavat solut rappeutuvat ja näköhermo vaurioituu. Kohonnut silmänpaine liittyy usein sairauteen. Väitöskirjan osatyöt keskittyivät verkkokalvon rappeumaan papillon ja phaléneja sekä länsigöötanmaanpystykorva (LJP) roduissa, sekä glaukoomaan dandiedinmontinterriereissä (DDT) ja harmaa norjanhirvikoirissa (HNH). Tutkimuksessa yhdistyy kansainvälinen kliinisen ja perustutkimuksen yhteistyö sairauksien diagnosoinnissa ja näytteiden keräämisessä, jossa hyödynnetään tutkimusryhmän ainutlaatuista yhteistyöverkkoa koirien omistajien ja kasvattajien kanssa. Merkittävien tutkimusresurssien rakentamisen lisäksi tutkimuksessa saavutettiin läpimurtoja kaikissa osaprojekteissa. Papillon ja phaléne rotujen verkkokalvonrappeuma johtuu geenivirheestä CNGB1 geenissä, joka liittyy vastaavaan sairauteen myös ihmisillä. LJP rodussa kuvattiin uudentyyppinen verkkokalvonrappeuma, joka liittyy MERTK geenin toimintahäiriöön. DDT rodun glaukooma paikannettiin kromosomialueelle, jonka vastaavalla alueella on useita glaukoomaan liitettyjä geenialueita ihmisellä. HNH rodun glaukooma liittyy puolestaan virheeseen ADAMTS10 geenissä. Kyseinen geeni aiheuttaa Weill-Marchesani syndroomaan (WMS) ihmisillä, jossa potilailla diagnosoidaan poikkeavuuksia silmissä ja mm. nivelissä. Teoreettisen ymmärryksen lisäksi tutkimuksella on tärkeitä käytännön vaikutuksia. Geenilöydöt korostavat ihmisen ja koirien silmäsairauksien yhteisiä perintötekijöitä ja tautimekanismeja. Tutkimus on myös paikantanut uusia malleja mahdollisiin terapiakokeiluihin. Geenilöydöt ovat tuoneet uusia työkaluja silmäsairauksien diagnostiikkaan ja ovat muuttaneet jalostuskäytäntöjä sairausgeenien karsimiseksi roduista geenitestien muodossa

A CNGB1 Frameshift Mutation in Papillon and Phalene Dogs with Progressive Retinal Atrophy

Peer reviewe

Reindeer grazing history determines the responses of subarctic soil fungal communities to warming and fertilization

Composition and functioning of arctic soil fungal communities may alter rapidly due to the ongoing trends of warmer temperatures, shifts in nutrient availability and shrub encroachment. In addition, the communities may also be intrinsically shaped by heavy grazing, which may locally induce an ecosystem change that couples with increased soil temperature and nutrients and where shrub encroachment is less likely to occur than in lightly grazed conditions. We tested how four years of experimental warming and fertilization affected organic soil fungal communities in sites with decadal history of either heavy or light reindeer grazing using high-throughput sequencing of ITS2 rDNA region. Grazing history largely overrode the impacts of short-term warming and fertilization in determining the composition of fungal communities. The less diverse fungal communities under light grazing showed more pronounced responses to experimental treatments when compared to the communities under heavy grazing. Yet, ordination approaches revealed distinct treatment responses under both grazing intensities. If grazing shifts the fungal communities in Arctic ecosystems to a different and more diverse state, this shift may dictate ecosystem responses to further abiotic changes. This inclines that the intensity of grazing cannot be left out when predicting future changes in fungi-driven processes in the tundra

Maternal Inheritance of a Recessive RBP4 Defect in Canine Congenital Eye Disease

Maternally skewed transmission of traits has been associated with genomic imprinting and oocytederived mRNA. We report canine congenital eye malformations, caused by an amino acid deletion (K12deI) near the N terminus of retinol-binding protein (RBP4). The disease is only expressed when both dam and offspring are deletion homozygotes. RBP carries vitamin A (retinol) from hepatic stores to peripheral tissues, including the placenta and developing eye, where it is required to synthesize retinoic acid. Gestational vitamin A deficiency is a known risk factor for ocular birth defects. The K12del mutation disrupts RBP folding in vivo, decreasing its secretion from hepatocytes to serum. The maternal penetrance effect arises from an impairment in the sequential transfer of retinol across the placenta, via RBP encoded by maternal and fetal genomes. Our results demonstrate a mode of recessive maternal inheritance, with a physiological basis, and they extend previous observations on dominant-negative RBP4 alleles in humans.Peer reviewe

Assessment of Canine \u3cem\u3eBEST1\u3c/em\u3e Variations Identifies New Mutations and Establishes an Independent Bestrophinopathy Model (\u3cem\u3ecmr3\u3c/em\u3e)

Purpose: Mutations in bestrophin 1 (BEST1) are associated with a group of retinal disorders known as bestrophinopathies in man and canine multifocal retinopathies (cmr) in the dog. To date, the dog is the only large animal model suitable for the complex characterization and in-depth studies of Best-related disorders. In the first report of cmr, the disease was described in a group of mastiff-related breeds (cmr1) and the Coton de Tulear (cmr2). Additional breeds, e.g., the Lapponian herder (LH) and others, subsequently were recognized with similar phenotypes, but linked loci are unknown. Analysis of the BEST1 gene aimed to identify mutations in these additional populations and extend our understanding of genotype–phenotype associations. Methods: Animals were subjected to routine eye exams, phenotypically characterized, and samples were collected for molecular studies. Known BEST1 mutations were assessed, and the canine BEST1 coding exons were amplified and sequenced in selected individuals that exhibited a cmr compatible phenotype but that did not carry known mutations. Resulting sequence changes were genotyped in several different breeds and evaluated in the context of the phenotype. Results: Seven novel coding variants were identified in exon 10 of cBEST1. Two linked mutations were associated with cmr exclusive to the LH breed (cmr3). Two individuals of Jämthund and Norfolk terrier breeds were heterozygous for two conservative changes, but these were unlikely to have disease-causing potential. Another three substitutions were found in the Bernese mountain dog that were predicted to have a deleterious effect on protein function. Previously reported mutations were excluded from segregation in these populations, but cmr1 was confirmed in another mastiff-related breed, the Italian cane corso. Conclusions: A third independent canine model for human bestrophinopathies has been established in the LH breed. While exhibiting a phenotype comparable to cmr1 and cmr2, the novel cmr3 mutation is predicted to be based on a distinctly different molecular mechanism. So far cmr2 and cmr3 are exclusive to a single dog breed each. In contrast, cmr1 is found in multiple related breeds. Additional sequence alterations identified in exon 10 of cBEST1 in other breeds exhibit potential disease-causing features. The inherent genetic and phenotypic variation observed with retinal disorders in canines is complicated further by cmr3 being one of four distinct genetic retinal traits found to segregate in LH. Thus, a combination of phenotypic, molecular, and population analysis is required to establish a strong phenotype–genotype association. These results indicate that cmr has a larger impact on the general dog population than was initially suspected. The complexity of these models further confirms the similarity to human bestrophinopathies. Moreover, analyses of multiple canine models will provide additional insight into the molecular basis underlying diseases caused by mutations in BEST1

Clinical and Genetic Findings in 28 American Cocker Spaniels with Aural Ceruminous Gland Hyperplasia and Ectasia

American Cocker Spaniels (ACSs) develop aural ceruminous gland hyperplasia and ectasia more often than dogs of other breeds. Data on the cause and development of these breed characteristic histopathological changes are lacking. We performed video-otoscopic examinations and dermatological work-up on 28 ACSs, obtained aural biopsies from each dog and assessed the statistical associations between the presence of ceruminous gland hyperplasia and ectasia and disease history, clinical or microbiological findings and underlying cause of otitis externa (OE). Histological lesions of ceruminous gland hyperplasia and ectasia were observed in aural biopsies from 6/13 clinically healthy ears and 13/15 ears with OE from 19/28 examined dogs. Nine of 28 dogs had histologically normal ceruminous glands (odds ratio [OR] 6.2, 95% confidence interval [CI] 1.1-36.6). Bacterial growth in microbiological culture of aural exudate (OR 14.1, 95% CI 2.1-95.3) was associated with ceruminous glandular changes, whereas previous history of OE, cutaneous findings or underlying allergies were not. Pedigree analysis and a genome-wide association study (GWAS) were performed on 18 affected and eight unaffected dogs based on histopathological diagnosis. While the GWAS indicated a tentative, but not statistically significant, association of ceruminous gland hyperplasia and ectasia with chromosome 31, a larger cohort is needed to confirm this preliminary result. Based on our results, ceruminous gland hyperplasia and ectasia may also precede clinical signs of OE in ACSs and a genetic aetiological component is likely Further studies with larger cohorts are warranted to verify our preliminary results. (C) 2021 The Authors. Published by Elsevier Ltd.Peer reviewe

Increased Expression of MERTK is Associated with a Unique Form of Canine Retinopathy

Progressive retinal degenerations are among the most common causes of blindness both in human and in dogs. Canine progressive retinal atrophy (PRA) resembles human retinitis pigmentosa (RP) and is typically characterized by a progressive loss of rod photoreceptors followed by a loss of cone function. The disease gradually progress from the loss of night and day vision to a complete blindness. We have recently described a unique form of retinopathy characterized by the multifocal gray/brown discoloration and thinning of the retina in the Swedish Vallhund (SV) breed. We aimed to identify the genetic cause by performing a genome wide association analysis in a cohort of 18 affected and 10 healthy control dogs using Illumina's canine 22k SNP array. We mapped the disease to canine chromosome 17 (p = 7.7×10−5) and found a 6.1 Mb shared homozygous region in the affected dogs. A combined analysis of the GWAS and replication data with additional 60 dogs confirmed the association (p = 4.3×10−8, OR = 11.2 for homozygosity). A targeted resequencing of the entire associated region in four cases and four controls with opposite risk haplotypes identified several variants in the coding region of functional candidate genes, such as a known retinopathy gene, MERTK. However, none of the identified coding variants followed a compelling case- or breed-specific segregation pattern. The expression analyses of four candidate genes in the region, MERTK, NPHP1, ANAPC1 and KRCC1, revealed specific upregulation of MERTK in the retina of the affected dogs. Collectively, these results indicate that the retinopathy is associated with overexpression of MERTK, however further investigation is needed to discover the regulatory mutation for the better understanding of the disease pathogenesis. Our study establishes a novel gain-of-function model for the MERTK biology and provides a therapy model for retinopathy MERTK inhibitors. Meanwhile, a marker-based genetic counseling can be developed to revise breeding programs.Peer reviewe

Canine MPV17 truncation without clinical manifestations

Mitochondrial DNA depletion syndromes (MDS) are often serious autosomal recessively inherited disorders characterized by tissue-specific mtDNA copy number reduction. Many genes, including MPV17, are associated with the hepatocerebral form of MDS. MPV17 encodes for a mitochondrial inner membrane protein with a poorly characterized function. Several MPV17 mutations have been reported in association with a heterogeneous group of early-onset manifestations, including liver disease and neurological problems. Mpv17-deficient mice present renal and hearing defects. We describe here a MPV17 truncation mutation in dogs. We found a 1-bp insertion in exon 4 of the MPV17 gene, resulting in a frameshift and early truncation of the encoded protein. The mutation halves MPV17 expression in the lymphocytes of the homozygous dogs and the truncated protein is not translated in transfected cells. The insertion mutation is recurrent and exists in many unrelated breeds, although is highly enriched in the Boxer breed. Unexpectedly, despite the truncation of MPV17, we could not find any common phenotypes in the genetically affected dogs. The lack of observable phenotype could be due to a late onset, mild symptoms or potential tissue-specific compensatory mechanisms. This study suggests species-specific differences in the manifestation of the MPV17 defects and establishes a novel large animal model to further study MPV17 function and role in mitochondrial biology.Peer reviewe

A Novel Missense Mutation in ADAMTS10 in Norwegian Elkhound Primary Glaucoma

The PLOS ONE Staff (2015) Correction: A Novel Missense Mutation in ADAMTS10 in Norwegian Elkhound Primary Glaucoma. PLoS ONE 10(2): e0118256. doi:10.1371/journal.pone.0118256Peer reviewe

An intronic LINE-1 insertion in MERTK is strongly associated with retinopathy in Swedish Vallhund dogs

The domestic dog segregates a significant number of inherited progressive retinal diseases, several of which mirror human retinal diseases and which are collectively termed progressive retinal atrophy (PRA). In 2014, a novel form of PRA was reported in the Swedish Vallhund breed, and the disease was mapped to canine chromosome 17. The causal mutation was not identified, but expression analyses of the retinas of affected Vallhunds demonstrated a 6-fold increased expression of the MERTK gene compared to unaffected dogs. Using 24 retinopathy cases and 97 controls with no clinical signs of retinopathy, we replicated the chromosome 17 association in Swedish Vallhunds from the UK and aimed to elucidate the causal variant underlying this association using whole genome sequencing (WGS) of an affected dog. This revealed a 6-8 kb insertion in intron 1 of MERTK that was not present in WGS of 49 dogs of other breeds. Sequencing and BLASTN analysis of the inserted segment was consistent with the insertion comprising a full-length intact LINE-1 retroelement. Testing of the LINE-1 insertion for association with retinopathy in the UK set of 24 cases and 97 controls revealed a strong statistical association (P-value 6.0 x 10(-11)) that was subsequently replicated in the original Finnish study set (49 cases and 89 controls (P-value 4.3 x 10(-19)). In a pooled analysis of both studies (73 cases and 186 controls), the LINE-1 insertion was associated with a similar to 20-fold increased risk of retinopathy (odds ratio 23.41, 95% confidence intervals 10.99-49.86, P-value 1.3 x 10(-27)). Our study adds further support for regulatory disruption of MERTK in Swedish Vallhund retinopathy; however, further work is required to establish a functional overexpression model. Future work to characterise the mechanism by which this intronic mutation disrupts gene regulation will further improve the understanding of MERTK biology and its role in retinal function.Peer reviewe