Two variants in the KIT gene as candidate causative mutations for a dominant white and a white spotting phenotype in the donkey

White spotting phenotypes have been intensively studied in horses, and although similar phenotypes occur in the donkey, little is known about the molecular genetics underlying these patterns in donkeys. White spotting in donkeys can range from only a few white areas to almost complete depigmentation and is characterised by a loss of pigmentation usually progressing from a white spot in the hip area. Completely white‐born donkeys are rare, and the phenotype is characterised by the complete absence of pigment resulting in pink skin and a white coat. A dominant mode of inheritance has been demonstrated for spotting in donkeys. Although the mode of inheritance for the completely white phenotype in donkeys is not clear, the phenotype shows similarities to dominant white in horses. As variants in the KIT gene are known to cause a range of white phenotypes in the horse, we investigated the KIT gene as a potential candidate gene for two phenotypes in the donkey, white spotting and white. A mutation analysis of all 21 KIT exons identified a missense variant in exon 4 (c.662A>C; p.Tyr221Ser) present only in a white‐born donkey. A second variant affecting a splice donor site (c.1978+2T>A) was found exclusively in donkeys with white spotting. Both variants were absent in 24 solid‐coloured controls. To the authors’ knowledge, this is the first study investigating genetic mechanisms underlying white phenotypes in donkeys. Our results suggest that two independent KIT alleles are probably responsible for white spotting and white in donkeys.     

A de novo mutation in KIT causes white spotting in a subpopulation of German Shepherd dogs

Although variation in the KIT gene is a common cause of white spotting among domesticated animals, KIT has not been implicated in the diverse white spotting observed in the dog. Here, we show that a loss‐of‐function mutation in KIT recapitulates the coat color phenotypes observed in other species. A spontaneous white spotting observed in a pedigree of German Shepherd dogs was mapped by linkage analysis to a single locus on CFA13 containing KIT (pairwise LOD = 15). DNA sequence analysis identified a novel 1‐bp insertion in the second exon that co‐segregated with the phenotype. The expected frameshift and resulting premature stop codons predicted a severely truncated c‐Kit receptor with presumably abolished activity. No dogs homozygous for the mutation were recovered from multiple intercrosses (P = 0.01), suggesting the mutation is recessively embryonic lethal. These observations are consistent with the effects of null alleles of KIT in other species.     

Hematologic parameters of the white donkeys of Asinara]

In 14 albino donkeys (6 males and 8 females) aged 1-12 years, about 25% of the whole population, 30 blood parameters were determined, with automatic equipment, monthly, for 4 months. No significant differences between sexes or compared with other donkey breeds were found, in spite of the particular characteristics of the population and of their environment.     

Identification of a novel nonsense mutation on the Pax3 gene in ENU-derived white belly spotting mice and its genetic interaction with c-Kit

In the course of a large-scale screening program of N-ethyl-N-nitrosourea mutagenesis, we isolated two semidominant mutation lines with white belly spotting, named as wps and wbs. Direct sequencing detected a nucleotide G-to-A transversion in exon 2 of the c-Kit gene in wps, which resulted in a missense D60N mutation. Another mutant, wbs, was mapped to chromosome 1 by genome-wide linkage analysis. In 93 meioses, the wbs locus was confined to a 5.2-Mb region between D1Mit380 and D1Mit215, including the Pax3 gene. A nonsense mutation K107X on the Pax3 coding region in wbs mice was identified, causing the loss of Pax3 protein in the homozygous mutant. We further demonstrated that Pax3 exhibited genetic interaction with c-Kit by intercrossing the wps and wbs mice. Further, Pax3 transactivated the c-Kit promoter in different cell lines. However, electrophoretic mobility shift assays showed that Pax3 did not bind to the c-Kit promoter, indicating that Pax3 may interact with c-Kit in an indirect way. This expands our understanding of the intricate regulatory network governing the melanocyte development.     

Clioquinol-zinc chelate: a candidate causative agent of subacute myelo-optic neuropathy.

BACKGROUND: 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol) was used clinically three decades ago as an oral antiparasitic agent and to increase intestinal absorption of zinc in patients with acrodermatitis enteropathica, a genetic disorder of zinc absorption. Use of clioquinol was epidemiologically linked to subacute myelo-optic neuropathy (SMON), characterized by peripheral neuropathy and blindness, which affected 10,000 patients in Japan. Discontinuation of oral clioquinol use led to elimination of SMON, however, the mechanism of how clioquinol induces neurotoxicity is unclear. MATERIALS AND METHODS: We tested the effect of clioquinol-metal chelates on neural crest-derived melanoma cells. The effect of clioquinol chelates on cells was further studied by electron microscopy and by a mitochondrial potential-sensitive fluorescent dye. RESULTS: Of the ions tested, only clioquinol-zinc chelate demonstrated cytotoxicity. The cytotoxicity of clioquinol-zinc chelate was extremely rapid, suggesting that its primary effect was on the mitochondria. Electron microscopic analysis demonstrated that clioquinol-zinc chelate caused mitochondrial damage. This finding was further confirmed by the observation that clioquinol-zinc chelate caused a decrease in mitochondrial membrane potential. CONCLUSIONS: We demonstrate that clioquinol, in the presence of zinc, is converted to a potent mitochondrial toxin. The phenomenon of clioquinol mediated toxicity appears to be specific to zinc and is not seen with other metals tested. Since clioquinol has been shown to cause increased systemic absorption of zinc in humans, it is likely that clioquinol-zinc chelate was present in appreciable levels in patients with SMON and may be the ultimate causative toxin of SMON.     

Exclusion of EDNRB and KIT as the basis for white spotting in Border Collies

Background  

White spotting patterns in mammals can be caused by mutations in the genes for the endothelin B receptor and c-Kit, whose protein products are necessary for proper migration, differentiation or survival of the melanoblast population of cells. Although there are many different dog breeds that segregate white spotting patterns, no genes have been identified that are linked to these phenotypes.     

Analysis of the inheritance of white spotting and the evaluation of KIT and EDNRB as spotting loci in Dutch boxer dogs

The genetic basis of the white spotting pattern in Dutch boxer dogs is not known. We studied whether the segregation of white spotting in boxers follows a Mendelian inheritance pattern. Blood samples were collected, along with digital photographs in standard directions of (grand)parents (n=16) and offspring (n=52) from eight litters of Dutch boxers. In order to select heterozygous parents, we selected nonuniform litters, in which at least one puppy was extreme white. On the basis of criteria for the location, the extent of white spotting, and the mean percentage of pigmented area of the foot soles, we classified 10 dogs as solid colored, 27 as flashy, and 15 as extreme white. This was not a significant deviation from the expected 1:2:1 ratio. Because the flashy phenotype seems to be an intermediate between the two homozygotes, white spotting in the Dutch boxer can be considered to be due to a single gene effect, with incomplete dominance. We have evaluated candidate genes c-KIT (KIT) and EDNRB for segregation with white spotting phenotype in these litters. Using polymorphic markers, very near the KIT and EDNRB genes, we found that segregation of the white spotting pattern did not coincide with segregation of these polymorphic markers. Thus neither KIT nor EDNRB are likely to be responsible for white spotting in the Dutch population of boxers.     

Mitochondrial mutation as a probable causative factor in familial progressive tubulointerstitial nephritis

Renal biopsy of two children and a maternal relative, diagnosed with severe progressive tubulointerstitial nephritis, has shown the presence of distorted mitochondria. Mitochondrial DNA from the blood of these patients was analysed. No major deletions were found, but an A to G mutation was detected in position 5656. It is proposed that this mutation might play a causative role in the renal disease of the patients. Received: 25 July 1996 / Revised: 15 October 1996     

Mutations in MITF and PAX3 cause "splashed white" and other white spotting phenotypes in horses

During fetal development neural-crest-derived melanoblasts migrate across the entire body surface and differentiate into melanocytes, the pigment-producing cells. Alterations in this precisely regulated process can lead to white spotting patterns. White spotting patterns in horses are a complex trait with a large phenotypic variance ranging from minimal white markings up to completely white horses. The "splashed white" pattern is primarily characterized by an extremely large blaze, often accompanied by extended white markings at the distal limbs and blue eyes. Some, but not all, splashed white horses are deaf. We analyzed a Quarter Horse family segregating for the splashed white coat color. Genome-wide linkage analysis in 31 horses gave a positive LOD score of 1.6 in a region on chromosome 6 containing the PAX3 gene. However, the linkage data were not in agreement with a monogenic inheritance of a single fully penetrant mutation. We sequenced the PAX3 gene and identified a missense mutation in some, but not all, splashed white Quarter Horses. Genome-wide association analysis indicated a potential second signal near MITF. We therefore sequenced the MITF gene and found a 10 bp insertion in the melanocyte-specific promoter. The MITF promoter variant was present in some splashed white Quarter Horses from the studied family, but also in splashed white horses from other horse breeds. Finally, we identified two additional non-synonymous mutations in the MITF gene in unrelated horses with white spotting phenotypes. Thus, several independent mutations in MITF and PAX3 together with known variants in the EDNRB and KIT genes explain a large proportion of horses with the more extreme white spotting phenotypes.     

Validation of a mobile phone-assisted microarray decoding platform for signal-enhanced mutation detection

We have established a mobile phone-assisted microarray decoding platform for signal-enhanced mutation detection. A large amount of single-stranded DNA (ssDNA) was obtained by combining symmetric PCR and magnetic isolation, and ssDNA prepared with magnetic bead as label was further allowed to hybridize against the tag-array for decoding purpose. High sensitivity and specificity was achieved with the detection of genomic DNA. When simultaneously genotyping nine common mutations associated with hereditary hearing loss, the detection limit of 1 ng genomic DNA was achieved. Significantly, a mobile phone was also used to record and decode the genotyping results through a custom-designed imaging adaptor and a dedicated mobile phone software. A total of 51 buccal swabs from patients probably with deafness-related mutations were collected and analyzed. The genotyping results were all confirmed by fluorescence-based laser confocal scanning and direct DNA sequencing. This mobile phone-assisted decoding platform provides an effective but economic mutation detection alternative for the future quicker and sensitive detection of virtually any mutation-related diseases in developing and underdeveloped countries.     

Polygalacturonase: a candidate gene for the soft flesh and deciduous fruit mutation in Capsicum

The soft flesh and deciduous fruit of pepper (Capsicum spp.) originated from the wild C. frutescens BG 2816 accession is a complete dominant trait controlled by the S gene. We constructed an F₂ population from a cross of BG 2816 (SS) and the bell-type C. annuum cultivar Maor (ss) and determined that S cosegregated with the tomato fruit-specific endo-polygalacturonase (PG) gene. The soft flesh and deciduous fruit phenotypes were observed together in all F₂ individuals, indicating a pleiotropic effect of PG on the two traits. We mapped S to pepper chromosome 10 in the region corresponding to that in which PG was previously mapped in tomato. Northern, RT-PCR and western analyses and enzyme activity assays, collectively, indicated that PG is not detected in green, breaker or red fruits of Maor, nor in green fruits of BG 2816. Accumulation of PG mRNA and protein was detected in the fruits of BG 2816, and it increased during ripening from breaker to red stages. The sequence analysis of partial PG cDNA isolated from BG 2816 revealed high homology (87% identity) with the tomato PG. The resemblance of the soft flesh and deciduous fruit phenotypes to PG-associated phenotypes in other fruit crops, the complete linkage between Sand PG, and the greater expression of PG in the fruits of BG 2816 than in those of Maor, all strongly indicate that PG is a candidate gene for S.     

The albinism of the feral Asinara white donkeys (Equus asinus) is determined by a missense mutation in a highly conserved position of the tyrosinase (TYR) gene deduced protein

A feral donkey population (Equus asinus), living in the Asinara National Park (an island north‐west of Sardinia, Italy), includes a unique white albino donkey subpopulation or colour morph that is a major attraction of this park. Disrupting mutations in the tyrosinase (TYR) gene are known to cause recessive albinisms in humans (oculocutaneous albinism Type 1; OCA1) and other species. In this study, we analysed the donkey TYR gene as a strong candidate to identify the causative mutation of the albinism of these donkeys. The TYR gene was sequenced from 13 donkeys (seven Asinara white albino and six coloured animals). Seven single nucleotide polymorphisms were identified. A missense mutation (c.604C>G; p.His202Asp) in a highly conserved amino acid position (even across kingdoms), which disrupts the first copper‐binding site (CuA) of functional protein, was identified in the homozygous condition (G/G or D/D) in all Asinara white albino donkeys and in the albino son of a trio (the grey parents had genotype C/G or H/D), supporting the recessive mode of inheritance of this mutation. Genotyping 82 donkeys confirmed that Asinara albino donkeys had genotype G/G whereas all other coloured donkeys had genotype C/C or C/G. Across‐population association between the c.604C>G genotypes and the albino coat colour was highly significant (P = 6.17E?18). The identification of the causative mutation of the albinism in the Asinara white donkeys might open new perspectives to study the dynamics of this putative deleterious allele in a feral population and to manage this interesting animal genetic resource.