Genetic characterization of congenital defects in dogs : caudal dysplasia, ectodermal dysplasia and mucopolysaccharidosis VII

Since the sequencing of the Canis lupus familiaris genome the dog has become a powerful tool for scientists. Selective breeding has created more than 400 different breeds each representing genetic isolates with breed-specific morphological and behavioral characteristics. Unique population history, available genealogical records, veterinary diagnostics and novel genomic tools greatly facilitate gene mapping studies in dogs. Given that over 600 genetic disorders have been described in dogs and that most of them are similar to human conditions, dogs have emerged as a clinically relevant model for human inherited disorders. This study explores the genetics of three different inherited developmental defects in dogs, caudal dysplasia, ectodermal dysplasia, and mucopolysaccharidosis VII, which all have counterparts in human. In this study, various clinical and pathological techniques have been used to characterize the phenotypes, and genetic methods such as genome-wide association studies and next-generation sequencing to resolve the genetics of the diseases. Moreover, functional studies in mice have been performed to explore the molecular role during embryonic development. The discoveries made here have established the affected breeds as models to further explore disease mechanisms and therapeutic methods, identified new disease pathways, and offered novel approaches for further developmental studies. Furthermore, this work has enabled the development of genetic tests for breeding purposes. Three different phenotypes have been investigated in this study. First, we studied genetics of caudal dysplasia, which in its mildest form is presenting as short-tail phenotype in dogs. A mutation in T (brachyury homolog) was earlier identified to cause this phenotype in Pembroke Welsh Corgis. Homozygous mutations of T in mouse result in severe caudal dysplasia and embryonic lethality suggesting an essential role for the T gene during mammalian development. The presence of the documented T mutation, c.189C>G, was investigated in 23 other breeds demonstrating that short-tailed dogs from 17 breeds were heterozygous for the mutation that associated completely with the phenotype. The homozygous mutation was suggested to be lethal, as no dogs homozygous for the mutation were found and an approximately 30% decrease was seen in the size of Swedish Vallhund litters when both parents were short-tailed. However, short-tailed dogs were found from six breeds that did not carry the known substitution or any other mutations in the T coding regions and therefore other genetic factors are yet to be discovered that affect the development of the posterior mesodermal region. The short-tailed dogs which do not have T mutation will serve as models in future studies to identify possible novel genetic factors for caudal dysplasia and related medical conditions. Second, a new gene was identified for a hairless phenotype and some of its upstream regulators were characterized. Hairless dog breeds show a breed characteristic which is in clinical terms an ectodermal dysplasia. In this study, the causative mutation for canine ectodermal dysplasia (CED) was sought and subsequently the function of this novel gene in the ectodermal organ development was explored. Accordingly, a genome-wide association study was performed and CED was mapped to dog chromosome 17. Haplotype association testing revealed a 160-kb haplotype, which was fine-mapped using three different breeds. The causative genetic mutation for CED was identified as a 7-bp duplication producing a frameshift and premature stop codon in a previously uncharacterized canine gene forkhead box protein I3 (FOXI3). The study provided a novel gene focus to aid research into ectodermal development. Therefore, a detailed expression pattern of murine Foxi3 during the development of the ectodermal organs was constructed and a series of tissue culture experiments and expression analyses with mouse embryos were performed to assess the function of Foxi3 in mammalian embryogenesis. The results suggest that Foxi3 regulates hair follicle and tooth formation as well as the development of mammary and salivary gland, nail, and eye. Ectodysplasin and activin A were identified as upstream regulators of Foxi3. Third, Brazilian Terriers with severe skeletal defects at early puppyhood were identified through information provided by breeders. Subsequently, a major aim of this work was to describe the clinical and pathological features of the syndrome and to identify its genetic cause. Clinicopathological examinations and pedigree analysis demonstrated that the affected puppies had a recessive spondyloepihyseal dysplasia. The disease locus was mapped to chromosome 6 and a mutation leading to pathogenic p.P289L change in a conserved functional domain of β-glucuronidase (GUSB) was identified. Elevated glycosaminoglycans were detected in urine and only a residual β-glucuronidase activity was observed in the serum of the affected dogs, which confirmed the pathogenity of the mutation. GUSB defects result in mucopolysaccharidosis VII (MPS VII) in several species and thus the mutation defined the syndrome as MPS VII in Brazilian Terriers. Overall, this study illustrates how unique morphological diversity and enriched genetic alterations in closed populations can be efficiently harnessed to gain new insights into developmental biology across species. For example, the identification of the CED mutation in FOXI3 revealed a completely novel gene with a previously unknown essential function in ectodermal development. This work has established several novel large animal models to further explore disease mechanisms and to develop therapeutic methods. Moreover, several new DNA tests have been developed for different breeds of dogs to eradicate or, to control better, the conditions through improved breeding plans. This will improve the welfare of our beloved pets.Koira on viime vuosien aikana saavuttanut vankan tassunsijan tieteellisenä tutkimuskohteena. Koiran geneettistä tutkimusta edesauttaa erikoinen populaatiohistoria, sisäsiittoisuuden aiheuttama tiettyjen geenimuotojen rikastuminen sekä runsas morfologisten piirteiden ja luonneominaisuuksien vaihtelu. Ihminen on jalostanut yli 400 eri rotua, joihin on rikastunut yhteensä yli 600 perinnöllistä sairautta, jotka muistuttavat vastaavia ihmissairauksia. Jokainen rotu edustaa geneettisesti eristäytynyttä populaatiota, jolla kullakin on omat rakenteelliset ja käytökselliset rotuominaisuudet sekä oma tautiperimä. ----- Väitöskirja käsittelee kolmea perinnöllistä koirissa esiintyvää sairautta: kaudaalidysplasiaa, ektodermaalista dysplasiaa ja mukopolysakkaridoosi VII:ää. Vastaavia sairauksia esiintyy myös ihmisellä. Kaudaalidysplasia ilmenee koirilla lievimmillään hännättömyytenä tai lyhyenä häntänä ns. töpöhäntänä. Tutkimuksessa löydettiin 17 uutta rotua, joissa vallitsevasti periytyvä lyhythäntäisyys johtuu heterotsygoottisesta mutaatiosta T-geenissä. Tulokset vahvistivat myös, että homotsygoottinen mutaatio aiheuttaa koirilla sikiöaikaisen tai syntymänjälkeisen kuoleman ja vakavan kaudaalidysplasian. Lisäksi löydettiin kuusi rotua, joilla lyhythäntäisyyteen ei löydetty syytä T-geenistä. Geneettisten syiden tunnistaminen näistä roduista saattaisi tuoda uutta tietoa kaudaalidysplasian synnystä ja ruumiin takaosien kehityksestä. Tutkimuksen toisessa osa-alueessa tutkittiin joillakin roduilla esiintyvää rotutyypillistä ominaisuutta, karvattomuutta, joka on lääketieteellisesti määriteltynä ektodermaalinen dysplasia. Ominaisuuden osoitettiin aiheutuvan mutaatiosta FOXI3-geenissä, joka oli koiralla vielä tuntematon geeni. Jatkotutkimuksissa Foxi3:n toimintaa selvitettiin hiiren avulla ja osoitettiin sen osallistuvan useiden eri elinten kehityksen säätelyyn (karvafollikkelit, hampaat, maito- ja sylkirauhaset, kynnet ja silmä). Geenilöydön pohjalta tunnistettiin siis täysin uusi ektodermaalisten elinten kehitykseen vaikuttava tekijä. Kolmannessa osatyössä tutkittiin tietyssä rodussa esiintyvää sairautta, jonka havaittiin tutkimusten perusteella periytyvän peittyvästi ja pääasiallisten muutosten olevan luuston kehityksessä. Geneettisten tutkimusten avulla sairauden syyksi tunnistettiin mutaatio beta-glukuronidaasientsyymiä koodaavassa geenissä (GUSB). Sairauden osoitettiin olevan vastaava kuin ihmisen mukopolysakkaridoosi VII. Tutkimus osoittaa, kuinka koiran morfologinen monimuotoisuus ja pieniin populaatioihin rikastuneet geenimuutokset edesauttavat geneettistä tutkimusta tarjoten uutta tietoa elimistön kehityksestä ja toiminnasta. Tutkimuksen pohjalta on kehitetty useita uusia geenitestejä jalostuksen apuvälineeksi monille eri roduille, mikä edesauttaa parhaan ystävämme hyvinvointia

"Missä kaikki on niin kuin kotona" : Nuorten kokemuksia perhekotielämästä

Tutkimuksen tarkoituksena oli selvittää Keski-Suomen alueen ammatillisiin perhekoteihin sijoitet-tujen nuorten kokemuksia perhekodissa kasvamisesta ja elämisestä. Tarkoitus oli myös selvittää millainen on nuorten mielestä ihanneperhekoti ja onko perhekotityössä nuorten näkökulmasta tarvetta muutoksille. Tutkimuksessa selvitettiin lisäksi nuorten suhteita perhekodin vanhempiin ja millaisia näkemyksiä nuorille on syntynyt kasvatuksesta ja mielenterveyden edistämisestä perhe-kodissa. Tutkimuksen kohdejoukon muodosti kaksikymmentä 12–20 -vuotiasta ammatilliseen perhekotiin sijoitettua nuorta. Tutkimusmenetelmä oli laadullinen ja aineistonkeruumenetelmänä käytettiin Internet -kyselyä. Lisäksi suoritettiin kaksi haastattelua kyselyssä vaillinaiseksi jääneitä teemoja syventämään. Aineiston analysointitapana käytettiin teemoittelua, jonka avulla tutkimusaineistosta nostettiin esiin tutkimuksen kannalta merkittävät asiat. Tulosten raportoinnissa käsiteltiin kyselyn ja haastattelujen vastauksista kerättyjä yhteenvetoja teema-alueittain. Keskeisimpänä tutkimustuloksena ilmeni, että nuoret olivat pääasiassa tyytyväisiä elämään perhe-kodissa ja he olivat sitä mieltä, että sijoitus oli ollut heille oikea ratkaisu. He omasivat hyvät ja lämpimät välit perhekodin vanhempiin sekä ymmärsivät perhekodin rajojen ja sääntöjen olevan olemassa heidän hyvinvointiaan turvaamaan. Tuloksista havaittiin, etteivät kaikki nuoret pysty puhumaan perhekodin vanhempien kanssa, vaan turvautuvat mieluummin ulkopuolisiin työnteki-jöihin tai pitävät asiat itsellään. Nuoret toivoivat perhekodin vanhemmilta ymmärrystä ja aikaa kuunnella. Tutkimus tuo uutta tietoutta ja pohdittavaa nuorten näkökulmasta heidän kanssaan työtä tekeville perhekodin vanhemmille ja muille työntekijöille.The purpose of the study was to investigate the experiences of adolescents living and growing up in professional foster homes in Central Finland. The aim was also to find out how these young people would describe their ideal foster home and if there was any need for changes concerning the work done in foster homes. Another goal was to examine the young people’s relationships to their foster parents and their thoughts about parenting and about the promotion of mental health in a foster home. The target group of this study was twenty adolescents between the age of 12 and 20 living in a foster home. A qualitative research method was used and the data was collected by using an inquiry on the Internet. In addition, two interviews were carried out in order to gain further depth in some of the themes in this research. For analyzing the material, themes was used in order to bring out the relevant aspects for the research. For reporting the results, summaries of the themes in the inquiry and the interviews were created. One of the most fundamental results of this research was that most foster youths were satisfied with their life. They also considered that the decision of placing them into a foster home had been the right one. In most cases the relationship to foster parents was described as warm. Furthermore, the reason for the existence of rules in the foster home was understood. It was also discovered that some of the respondents found it easier to communicate with outside workers rather than with their foster parents. This research brings new information on the opinions of foster youths to foster parents and other people working with them

A frameshift insertion in SGK3 leads to recessive hairlessness in Scottish Deerhounds : a candidate gene for human alopecia conditions

Hairlessness is a breed-specific feature selected for in some dog breeds but a rare abnormality in some others such as Scottish Deerhounds (SD). In SDs, the affected puppies are born with sparse hair but lose it within the first 2months leaving the dogs completely hairless. The previous studies have implicated variants in FOXI3 and SGK3 in hairlessness; however, the known variants do not explain hairlessness in all breeds such as SDs. We investigated the genetic cause in 66 SDs, including a litter with two hairless dogs. We utilized a combined approach of genome-wide homozygosity mapping and whole-genome sequencing of a hairless SD followed by recessive filtering according to a recessive model against 340 control genomes. Only two homozygous-coding variants were discovered in the homozygosity regions, including a 1-bp insertion in exon 2 of SGK3. This results in a predicted frameshift and very early truncation (49/490 amino acids) of the SGK3 protein. Additional screening of the recessive variant demonstrated a full segregation with the hairlessness and a 12% carrier frequency in the SD breed. The variant was not found in the related Irish Wolfhound breed. This study identifies the second hairless variant in the SGK3 gene in dogs and further highlights its role as a candidate gene for androgen-independent hair loss or alopecia in human.Peer reviewe

Multi-omic analyses in Abyssinian cats with primary renal amyloid deposits

The amyloidoses constitute a group of diseases occurring in humans and animals that are characterized by abnormal deposits of aggregated proteins in organs, affecting their structure and function. In the Abyssinian cat breed, a familial form of renal amyloidosis has been described. In this study, multi-omics analyses were applied and integrated to explore some aspects of the unknown pathogenetic processes in cats. Whole-genome sequences of two affected Abyssinians and 195 controls of other breeds (part of the 99 Lives initiative) were screened to prioritize potential disease-associated variants. Proteome and miRNAome from formalin-fixed paraffin-embedded kidney specimens of fully necropsied Abyssinian cats, three affected and three non-amyloidosis-affected were characterized. While the trigger of the disorder remains unclear, overall, (i) 35,960 genomic variants were detected; (ii) 215 and 56 proteins were identified as exclusive or overexpressed in the affected and control kidneys, respectively; (iii) 60 miRNAs were differentially expressed, 20 of which are newly described. With omics data integration, the general conclusions are: (i) the familial amyloid renal form in Abyssinians is not a simple monogenic trait; (ii) amyloid deposition is not triggered by mutated amyloidogenic proteins but is a mix of proteins codified by wild-type genes; (iii) the form is biochemically classifiable as AA amyloidosis.Peer reviewe

Canine models of human rare disorders

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Koirien geenitutkimus edistää harvinaissairauksien ymmärtämistä

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webGQT: A Shiny Server for Genotype Query Tools for Model-Based Variant Filtering

Genotype Query Tools (GQT) were developed to discover disease-causing variations from billions of genotypes and millions of genomes, processes data at substantially higher speed over other existing methods. While GQT has been available to a wide audience as command-line software, the difficulty of constructing queries among non-IT or non-bioinformatics researchers has limited its applicability. To overcome this limitation, we developed webGQT, an easy-to-use tool with a graphical user interface. With pre-built queries across three modules, webGQT allows for pedigree analysis, case-control studies, and population frequency studies. As a package, webGQT allows researchers with less or no applied bioinformatics/IT experience to mine potential disease-causing variants from billions. Results webGQT offers a flexible and easy-to-use interface for model-based candidate variant filtering for Mendelian diseases from thousands to millions of genomes at a reduced computation time. Additionally, webGQT provides adjustable parameters to reduce false positives and rescue missing genotypes across all modules. Using a case study, we demonstrate the applicability of webGQT to query non-human genomes. In addition, we demonstrate the scalability of webGQT on large data sets by implementing complex population-specific queries on the 1000 Genomes Project Phase 3 data set, which includes 8.4 billion variants from 2504 individuals across 26 different populations. Furthermore, webGQT supports filtering single-nucleotide variants, short insertions/deletions, copy number or any other variant genotypes supported by the VCF specification. Our results show that webGQT can be used as an online web service, or deployed on personal computers or local servers within research groups. Availability webGQT is made available to the users in three forms: 1) as a webserver available at https://vm1138.kaj.pouta.csc.fi/webgqt/, 2) as an R package to install on personal computers, and 3) as part of the same R package to configure on the user's own servers. The application is available for installation at https://github.com/arumds/webgqt.Peer reviewe

Canine models of human amelogenesis imperfecta: identification of novel recessive ENAM and ACP4 variants

Amelogenesis imperfecta (AI) refers to a genetically and clinically heterogeneous group of inherited disorders affecting the structure, composition, and quantity of tooth enamel. Both non-syndromic and syndromic forms of AI have been described and several genes affecting various aspects of the enamel physiology have been reported. Genetically modified murine models of various genes have provided insights into the complex regulation of proper amelogenesis. Non-syndromic AI occurs spontaneously also in dogs with known recessive variants in ENAM and SLC24A4 genes. Unlike rodents with a reduced dentition and continuously erupting incisors, canine models are valuable for human AI due to similarity in the dental anatomy including deciduous and permanent teeth. We have performed a series of clinical and genetic analyses to investigate AI in several breeds of dogs and describe here two novel recessive variants in the ENAM and ACP4 genes. A fully segregating missense variant (c.716C>T) in exon 8 of ENAM substitutes a well-conserved proline to leucine, p.(Pro239Leu), resulting in a clinical hypomineralization of teeth. A 1-bp insertion in ACP4 (c.1189dupG) is predicted to lead to a frameshift, p.(Ala397Glyfs), resulting in an abnormal C-terminal part of the protein, and hypoplastic AI. The ENAM variant was specific for Parson Russell Terriers with a carrier frequency of 9%. The ACP4 variant was found in two breeds, Akita and American Akita with a carrier frequency of 22%. These genetic findings establish novel canine models of human AI with a particular interest in the case of the ACP4-deficient model, since ACP4 physiology is poorly characterized in human AI. The affected dogs could also serve as preclinical models for novel treatments while the breeds would benefit from genetic tests devised here for veterinary diagnostics and breeding programs.Peer reviewe

Myotonia congenita in a Labrador Retriever with truncated CLCN1

An eight week old Labrador Retriever puppy presented with stiff-legged robotic gait. Abnormal gait was most evident after rest and improved with prolonged activity. On occasions, initiation of sudden movements would result in collapse with rigidity of the trunk and stiff extended limbs for several seconds. Other clinical signs were excitement-induced upper airway stridor and oropharyngeal dysphagia. Myotonia congenita was diagnosed based on clinical signs, abundant myotonic discharges on electromyography and exclusion of structural myopathies on histology. Whole exome sequencing revealed a case-specific homozygous variant in CLCN1, c.2275A > T resulting in a premature stop codon, p.R759X. The CLCN1 variant was absent from the genomes of 127 Labrador Retriever controls and 474 control dogs from other breeds. This study expands the spectrum of identified canine CLCN1 mutations and the list of affected breeds in myotonia congenita and highlights the potential value of dogs as translational large animal models of human genetic diseases. (C) 2018 The Authors. Published by Elsevier B.V.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