Genetic investigation of spontaneous harlequin coat patterning in a family of Finnish Collies

The merle coat pattern is a pigmentary phenotype of dogs characterized by a dilute background with black patches. Merle is caused by a SINE insertion in PMEL17, a pigmentation gene expressed in melanocytes. The mutation causes aberrant splicing of transcripts and production of an abnormal protein. Harlequin is a dominant modifier of merle that further dilutes the background to white. Harlequin Great Danes have a heterozygous mutation impairing the ubiquitin-proteasome system, suggesting that the inability to degrade aberrant PMEL17 results in melanocyte death. Harlequin is not a recognized coat pattern of the Collie; however, a harlequin phenotype spontaneously appeared in a family of Finnish Collies. Pedigree analysis revealed an inheritance pattern consistent with a dominant modifier of merle occurring de novo in the proband. To identify the mutation, we generated 30X whole genome resequencing data from a second generation harlequin Collie. We identified over two million heterozygous variants and filtered for unique coding variants against 1400 canine genomes aligned to the CanFam3 reference genome. The remaining 155 variants were manually inspected in IGV revealing 10 candidate missense mutations. Sanger sequencing in family members revealed that none of the identified variants segregated with the harlequin phenotype. Future efforts will utilize newly available reference genomes to identify additional coding variants. The identification of novel mutations that cause harlequin patterning in merle dogs will provide insight into genes and sequences critical for proper functioning of the ubiquitin-proteasome system

A missense mutation in PMEL17 is associated with the Silver coat color in the horse

BACKGROUND: The Silver coat color, also called Silver dapple, in the horse is characterized by dilution of the black pigment in the hair. This phenotype shows an autosomal dominant inheritance. The effect of the mutation is most visible in the long hairs of the mane and tail, which are diluted to a mixture of white and gray hairs. Herein we describe the identification of the responsible gene and a missense mutation associated with the Silver phenotype. RESULTS: Segregation data on the Silver locus (Z) were obtained within one half-sib family that consisted of a heterozygous Silver colored stallion with 34 offspring and their 29 non-Silver dams. We typed 41 genetic markers well spread over the horse genome, including one single microsatellite marker (TKY284) close to the candidate gene PMEL17 on horse chromosome 6 (ECA6q23). Significant linkage was found between the Silver phenotype and TKY284 (θ = 0, z = 9.0). DNA sequencing of PMEL17 in Silver and non-Silver horses revealed a missense mutation in exon 11 changing the second amino acid in the cytoplasmic region from arginine to cysteine (Arg618Cys). This mutation showed complete association with the Silver phenotype across multiple horse breeds, and was not found among non-Silver horses with one clear exception; a chestnut colored individual that had several Silver offspring when mated to different non-Silver stallions also carried the exon 11 mutation. In total, 64 Silver horses from six breeds and 85 non-Silver horses from 14 breeds were tested for the exon 11 mutation. One additional mutation located in intron 9, only 759 bases from the missense mutation, also showed complete association with the Silver phenotype. However, as one could expect to find several non-causative mutations completely associated with the Silver mutation, we argue that the missense mutation is more likely to be causative. CONCLUSION: The present study shows that PMEL17 causes the Silver coat color in the horse and enable genetic testing for this trait

Equine trait mapping

Assigning function to genes is essential for a better understanding of biological systems. To date, approximately half of the genes in the vertebrate genome have known function. Domestic animals are a rich source for trait mapping and in this thesis we have mapped three distinct equine phenotypes. The result provides increased knowledge regarding gene function and importantly, practical implications for horse welfare. In paper I and IV, we confirm that Equine Multiple Congenital Ocular Anomalies (MCOA) syndrome is inherited as an incompletely dominant trait (p= 2.2x10-16). By first identifying a 208 kb identity-by decent (IBD) region and subsequently excluding polymorphic sites identified through Illumina sequencing, we conclude that the gene PMEL causes these defects in horse. Our findings, together with functional analyses recently published, support that the cause of MCOA syndrome is a missense mutation (Arg625Cys) near the transmembrane region of PMEL that results in altered biochemical properties. In paper II we show that variants in the MHC-II region influence the susceptibility to equine Insect Bite Hypersensitivity with the same marker risk allele identified in two distinct populations, OR 4.19 (p= 2.3x10-5) and 1.48 (p= 0.04) for Icelandic horses and Exmoor ponies respectively. In addition, homozygosity across the MHC-II region confers a higher risk of developing disease, OR= 2.67 (p= 1.3x10-3). Finally, in paper III we utilize the EquineSNP50 BeadChip to identify the first Gait locus in horse. A highly significant SNP (EMP2= 2.0x10-4) was identified to be consistent with a recessive mode of inheritance for the lateral gait pace in Icelandic horses, and confirmed in an independent sample set (p= 2.4x10-14). Illumina sequencing of an established IBD region identified a nonsense mutation in the gene DMRT3. A clearly dichotomous distribution in a panel of gaited and non-gaited breeds revealed that the DMRT3 mutation is permissive for a variety of alternate gaits. The mutation also has a favorable effect in harness racing horses. Functional characterization of the truncated protein demonstrated correct localization and an intact DNA binding profile. mRNA expression in a small population of commissural neurons from the spinal cord was confirmed in mutant and wild type horses. Further, a DMRT3 null mouse displayed a change in spinal cord circuit signaling and locomotion. These findings reveal a new molecule involved in the regulation of limb movement

Genetics of merle patterning in the domestic dog and gene transcript profiling and immunobiology of dermatomyositis in the shetland sheepdog

Since its domestication, the dog has served in many roles, from protector, guide, hunter, and best friend, to model organism. Every role in which the dog serves is important; however, this work highlights the importance of the dog as a model organism for study of human hereditary diseases. Roughly half of the 450 hereditary diseases found in the dog have clinical presentations similar to those found in the human. Included in these are auditory-pigmentation conditions and skin diseases for which the dog is a working model. Described herein are studies of the merle coat pattern and dermatomyositis. Through research on these topics, important information can be obtained that can be used to help both the dog and the human. Merle is a pattern of coloring observed in the coat of the domestic dog and is characterized by patches of diluted pigment. Dogs heterozygous or homozygous for the merle locus exhibit a wide range of auditory and ophthalmologic abnormalities. Linkage disequilibrium was identified for a microsatellite marker with the merle phenotype in the Shetland Sheepdog. This region of the human genome contains SILV, a gene important in mammalian pigmentation. Therefore, this gene was evaluated as a candidate for merle patterning. A short interspersed element insertion at the boundary of intron 10/exon 11 was found, and this insertion segregates with the merle phenotype in multiple breeds. These data show that SILV is responsible for merle patterning and is associated with impaired function of the auditory and ophthalmologic systems. Dermatomyositis (DM) is an inflammatory disease of the skin and muscle that occurs most often in the rough collie and Shetland Sheepdog. Gene transcript profiles were generated for affected and normal skin using a canine-specific oligonucleotide array. Two-hundred and eight-five gene transcripts, many of which are involved in immune function, were found to be differentially regulated in these tissues. Also reported are western blot, immunohistochemistry, and immunofluorescence analyses. While our work suggests that canine DM is a disease that may be immune mediated, it did not detect the production of specific disease-associated autoantibodies

The cloning and characterisation of the chicken tyrosinase-related protein gene family

Very little is known about the molecular and genetic mechanisms controlling pigmentation within the bird kingdom. The aim therefore, of this study was to contribute towards the understanding of the genetic regulation of avian pigmentation by the cloning and characterisation of the chicken Tyrosinase-related protein (TRP) gene family. To accomplish this goal, neural crest cells from 500 black chick embryos were cultured under conditions supportive of melanocyte differentiation and proliferation. Using RNA extracted from these pigmented melanocyte cultures, a novel embryonic chick cDNA library was constructed. Screening of this library for chicken equivalents of the mammalian TRP gene family yielded more than 200 cDNA clones. After sequencing, three of these clones, 88.3, pcTRP- 1.6 and pcTRP -2. 10, were found to encode chicken Tyrosinase (Tyr), Tyrosinase-related protein-1 (Tyrp1) and Tyrosinase-related protein-2 (Tyrp2), respectively. In addition, a chicken Microphthalmia (Mi) cDNA clone (M156) was isolated using a mouse Mi cDNA probe. Comparative analyses revealed that chicken Tyr, Tyrp1 and Tyrp2 share approximately 68%, 72% and 70% amino acid sequence identity with their vertebrate orthologues. Northern blot hybridisation analysis demonstrated that the chicken TRPs are expressed in RNA from cultured retinal pigment epithelial (RPE) cells as well as in whole eye RNA. The major transcript sizes for the chicken Tyr, Tyrp1 and Tyrp2 genes are 2.5 kb, 2.3 kb and 3.5 kb, respectively. In situ hybridisation studies confirmed that both chicken Tyr and Tyrp2 genes are expressed in a pigment cell-specific fashion with signals detected in both the skin and RPE of chick embryos. Genomic Southern blot hybridisation analyses strongly suggested that all three chicken TRP genes contain several introns that are likely to be conserved within the vertebrate TRP gene family. Furthermore, the chick Tyr, Tyrp1 and Tyrp2 genes were found to span approximately 5-19 kb, 5-11 kb and 15-30 kb, respectively of the chicken genome. Comparisons between a black and white chick breed at the Tyr and Tyrp1 loci revealed no gross rearrangements at either of these loci. However, 1-2 kb alterations were observed between the same breeds at the Tyrp2 locus. The nature and significance of this alteration is not known. The cloning of the chicken Tyr, Tyrp1 and Tyrp2 cDNAs constitutes the first molecular cloning and characterisation of any avian TRP gene family. Taken together therefore, this study contributes towards the further understanding of the molecular mechanisms regulating pigmentation as well as the evolution of gene families

Novel vaccination strategies for CD4+ T cell immunotherapy of melanoma

Immunotherapy has emerged as a standard treatment modality in melanoma and many other cancers. While a lot is known about the anti-tumoral effector functions of CD8+ T cells, CD4+ T cells remain less well understood in cancer immunotherapy. In the current work, it was hypothesized that melanocyte antigen-specific CD4+ T cells can control the growth of melanomas as efficiently as corresponding CD8+ T cells but differ in the way they recognize antigen and exert their effector functions against tumor cells in the tissue microenvironment. It has been previously shown by the Tüting lab that a single administration of an adenovirus vector expressing the melanocytic antigen gp100 can promote effective expansion of adoptively transferred gp100-specific Pmel-1 TCR transgenic CD8+ T cells and cause regression of established melanomas in syngeneic mice. Here, a similar therapy protocol was established for Trp1-specific TCR transgenic CD4+ T cells. For this, the adenoviral vaccine vector Ad-GTY expressing both gp100 and Trp1 epitopes was first generated. Ad-GTY could expand adoptively transferred Trp1 CD4+ T cells in vivo, albeit less efficiently when compared to Pmel-1 CD8+ T cells. Nevertheless, a Trp1 CD4+ T cell ACT protocol with Ad-GTY showed significant anti-tumor efficacy and could control the growth of HCmel12 melanomas. The recombinant MVA virus vector MVA-PMTP that also expressed both the gp100 and Trp1 epitopes was generated to evaluate prime-boost vaccine strategies. However, MVA-PMTP was only able to re-expand CD8+ T cells but not CD4+ T cells. Moreover, the Ad-MVA prime-boost vaccination strategy did not significantly increase the therapeutic efficacy of the ACT protocols. Following Trp1 CD4+ ACT escaping melanoma cells frequently down-regulated melanocytic antigen expression and acquired a dedifferentiated phenotype presumably due to therapy-induced inflammation. As shown previously with CD8+ T cells, this also represented a major limitation of targeting melanocytic antigens with antigen-specific CD4+ T cells. Experiments using HCmel12 Trp1 antigen loss variants generated with CRISPR-Cas9 genome editing techniques revealed that the control of tumor growth by Trp1 CD4+ T cells is antigen-specific. Experiments with mixtures of HCmel12 control and Trp1 knockout cells demonstrated that Trp1 CD4+ cells can exert significant bystander killing and that immunoselection for irreversible genetic antigen loss is dominant over reversible phenotypic antigen loss for immune escape of melanoma cells. HCmel12 Ciita loss variants were also generated with CRISPR-Cas9 genome editing techniques. Unlike unmodified HCmel12 cells they fail to upregulate MHC class II and therefore cannot be directly recognized by Trp1 CD4+ T cells. Experiments revealed that direct MHC class II restricted recognition of melanoma cells by Trp1 CD4+ T cells was not required for tumor growth control in vivo. This suggested an important role for indirect stimulation of Trp1 CD4+ T cells by APC in the tumor microenvironment. Likely, Trp1 CD4+ T cells indirectly control melanoma growth in the tumor microenvironment through Th1 associated cytokines such as IFN-γ and TNF-α. Future studies will have to address the spatial location of Trp1 CD4+ T cells in the tumor microenvironment, their interaction with other immune cells and the role of Th1-associated cytokines for their anti-tumor efficacy. Combining T cell therapies with signal transduction inhibitors or checkpoint inhibitors to counteract mechanisms of therapy resistance and immune escape in mouse models will help to delineate strategies for more effective treatment of melanoma patients that should be tested in the clinic

A comprehensive review of mammalian pigmentation: paving the way for innovative hair colour-changing cosmetics

The natural colour of hair shafts is formed at the bulb of hair follicles, and it is coupled to the hair growth cycle. Three critical processes must happen for efficient pigmentation: (1) melanosome biogenesis in neural crest-derived melanocytes, (2) the biochemical synthesis of melanins (melanogenesis) inside melanosomes, and (3) the transfer of melanin granules to surrounding pre-cortical keratinocytes for their incorporation into nascent hair fibres. All these steps are under complex genetic control. The array of natural hair colour shades are ascribed to polymorphisms in several pigmentary genes. A myriad of factors acting via autocrine, paracrine, and endocrine mechanisms also contributes for hair colour diversity. Given the enormous social and cosmetic importance attributed to hair colour, hair dyeing is today a common practice. Nonetheless, the adverse effects of the long-term usage of such cosmetic procedures demand the development of new methods for colour change. In this context, case reports of hair lightening, darkening and repigmentation as a side-effect of the therapeutic usage of many drugs substantiate the possibility to tune hair colour by interfering with the biology of follicular pigmentary units. By scrutinizing mammalian pigmentation, this review pinpoints key targetable processes for the development of innovative cosmetics that can safely change the hair colour from the inside out.The authors thank the support of the Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of the UIDB/04469/2020 unit, and LABBELS— Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/ 0029/2020. The author Bruno Fernandes also acknowledges his PhD scholarship funded by FCT (SFRH/BD/131824/2017).info:eu-repo/semantics/publishedVersio