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