Mapping trait genes in dogs

Understanding the genetic background of a given phenotypic trait or disease has long intrigued scientists. Model organisms can be employed when it is not feasible or possible to find trait causality in human cohorts. This thesis reports the mapping of both a phenotypic trait (white spotting) and a disease (sensory ataxic neuropathy) in dogs, and in doing so highlights the potential of the dog as a model organism for mapping traits of relevance to human health and biology. For white spotting, a two-stage mapping approach was used to identify an associated genomic region, which contained only the microphtalmia-associated transcription factor (MITF) gene. Stage 1: a genome-wide association analysis in a single breed, taking advantage of the extensive within breed linkage disequilibrium (LD) and long haplotypes, identified a discrete region of approximately 1 Mb. Stage 2: finemapping in an additional breed presenting the same phenotype, exploiting the short LD and haplotypes shared across dog breeds, was used to narrow the region to about 100 kb. We functionally evaluated two candidate polymorphisms associated with MITF, a SINE insertion and a length polymorphism upstream of the melanocyte-specific transcription start site of MITF. The data indicated that both polymorphisms affect transcription from the MITF-M promoter. Sensory ataxic neuropathy (SAN) is a neurological disorder affecting a specific maternal lineage of Golden Retrievers. We identified a one base pair deletion in mitochondrial tRNATyr and through biochemical analyses of mitochondria and functional studies of the deletion, confirmed causality and the mitochondrial origin of SAN. This is one of the first mitochondrial disorders identified in dogs and we additionally developed a genetic test for the public to allow for the elimination of the disease from this breed. The genetic and functional analyses of both white spotting coat colour and SAN in dogs, which are controlled by mutations of the two different genomes, clearly demonstrate the utility of the canine model and establish a new role for man's best friend

A progressive and complex clinical course in two family members with ERF-related craniosynostosis : a case report

Background: ERF-related craniosynostosis are a rare, complex, premature trisutural fusion associated with a broad spectrum of clinical features and heterogeneous aetiology. Here we describe two cases with the same pathogenic variant and a detailed description of their clinical course. Case presentation: Two subjects; a boy with a BLSS requiring repeated skull expansions and his mother who had been operated once for sagittal synostosis. Both developed intracranial hypertension at some point during the course, which was for both verified by formal invasive intracranial pressure monitoring. Exome sequencing revealed a pathogenic truncating frame shift variant in the ERF gene. Conclusions: Here we describe a boy and his mother with different craniosynostosis patterns, but both with verified intracranial hypertension and heterozygosity for a truncating variant of ERF c.1201_1202delAA (p.Lys401Glufs*10). Our work provides supplementary evidence in support of previous phenotypic descriptions of ERF-related craniosynostosis, particularly late presentation, an evolving synostotic pattern and variable expressivity even among affected family members

A Simple Repeat Polymorphism in the MITF-M Promoter Is a Key Regulator of White Spotting in Dogs

The white spotting locus (S) in dogs is colocalized with the MITF (microphtalmia-associated transcription factor) gene. The phenotypic effects of the four S alleles range from solid colour (S) to extreme white spotting (sw). We have investigated four candidate mutations associated with the sw allele, a SINE insertion, a SNP at a conserved site and a simple repeat polymorphism all associated with the MITF-M promoter as well as a 12 base pair deletion in exon 1B. The variants associated with white spotting at all four loci were also found among wolves and we conclude that none of these could be a sole causal mutation, at least not for extreme white spotting. We propose that the three canine white spotting alleles are not caused by three independent mutations but represent haplotype effects due to different combinations of causal polymorphisms. The simple repeat polymorphism showed extensive diversity both in dogs and wolves, and allele-sharing was common between wolves and white spotted dogs but was non-existent between solid and spotted dogs as well as between wolves and solid dogs. This finding was unexpected as Solid is assumed to be the wild-type allele. The data indicate that the simple repeat polymorphism has been a target for selection during dog domestication and breed formation. We also evaluated the significance of the three MITF-M associated polymorphisms with a Luciferase assay, and found conclusive evidence that the simple repeat polymorphism affects promoter activity. Three alleles associated with white spotting gave consistently lower promoter activity compared with the allele associated with solid colour. We propose that the simple repeat polymorphism affects cooperativity between transcription factors binding on either flanking sides of the repeat. Thus, both genetic and functional evidence show that the simple repeat polymorphism is a key regulator of white spotting in dogs.Peer reviewe

WNT3 involvement in human bladder exstrophy and cloaca development in zebrafish.

Bladder exstrophy, a severe congenital urological malformation when a child is born with an open urinary bladder, is the most common form of bladder exstrophy-epispadias complex (BEEC) with an incidence of 1:30.000 children of Caucasian descent. Recent studies suggest that WNT genes may contribute to the etiology of bladder exstrophy. Here, we evaluated WNT pathway genes in 20 bladder exstrophy patients using massively parallel sequencing. In total 13 variants were identified in WNT3, WNT6, WNT7A, WNT8B, WNT10A, WNT11, WNT16, FZD5, LRP1 and LRP10 genes and predicted as potentially disease causing, of which seven variants were novel. One variant, identified in a patient with a de novo nonsynonymous substitution in WNT3 (p.Cys91Arg), was further evaluated in zebrafish. Knock down of wnt3 in zebrafish showed cloaca malformations, including disorganization of the cloaca epithelium and expansion of the cloaca lumen. Our study suggests that the function of the WNT3 p.Cys91Arg variant was altered, since RNA overexpression of mutant Wnt3 RNA does not result in embryonic lethality as seen with wild type WNT3 mRNA. Finally, we also mutation screened the WNT3 gene further in 410 DNA samples from BEEC cases and identified one additional mutation c.638G>A (p.Gly213Asp), which was paternally inherited. In aggregate our data support the involvement of WNT pathway genes in BEEC and suggest that WNT3 in itself is a rare cause of BEEC

An example of interictal EEG recording for an epileptic (A) and for a healthy (B) BS dog.

<p>The epileptic and healthy dogs correspond to dogs 26 and 4C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033549#pone-0033549-t001" target="_blank">Table 1</a>, respectively. The Epileptic dog shows spike and slow waves in right central and posterior derivations. The control dog exhibits a high-voltage low-frequency background activity. Background activity is superimposed with focal beta bursts in frontal derivation. The EEG pattern is consistent with the sedation protocol used. Bipolar montage, time constant = 0.3 s; high filter 70 Hz; notch filter inserted.</p

Summary of clinical examinations performed on 17 epileptic and 4 healthy Finnish BS dogs.

1<p>CFG = complex focal generalized, CF = complex focal, GUO = generalized with unknown onset, NA = not available.</p>2<p>IE = idiopathic epilepsy.</p