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

Intravenöse und perorale Behandlung von Kühen mit Gebärparese mit Kalzium und Natriumphosphat

Bei Kühen mit Gebärparese wurden der Elektrolytverlauf und der Therapieerfolg nach Behandlung mit Kalziumboroglukonat intravenös bzw. nach Kalziumboroglukonat und Natriumdihydrogenphosphat intravenös sowie Kalziumlaktat und Natriumhydrogenphosphat peroral untersucht. 30 Kühe mit Gebärparese wurden in zwei Gruppen à je 15 Tiere eingeteilt. Den Kühen beider Gruppen wurden 500 ml einer 40%igen Kalziumboroglukonatlösung verabreicht. Die Kühe der Versuchsgruppe wurden zudem mit 500 ml einer Natriumdihydrogenphosphat Lösung intravenös sowie mit Kalziumlaktat und Natriumhydrogenphosphat peroral behandelt. Danach wurden die Kalzium, Phosphor- und Magnesiumverlaufskurven und das Ansprechen der Tiere auf die Therapie untersucht. Unabhängig von der Therapie stieg die Kalziumkonzentration nach der Behandlung vorübergehend stark an und sank dann langsam wieder ab. Die Phosphatkonzentrationen stiegen in der Kontrollgruppe, welche nur Kalzium erhalten hatte, kaum an, während es bei der mit Natriumphosphat behandelte Versuchsgruppe zu einem starken Anstieg kam. Der Therapieerfolg der beiden Gruppen unterschied sich nicht signifikant. Die Rezidivrate war jedoch bei der Versuchsgruppe signifikant höher als bei der Kontrollgruppe. In cows with parturient paresis the electrolyte concentrations and the therapeutic success after treatment with calciumborogluconate intravenously and after treatment with calciumborogluconate and sodium phosphate intravenously and calcium lactate and sodium phosphate orally respectively were examined. 30 cows with parturient paresis were divided into two groups of 15 animals each. The cows of both groups were treated with 500 ml of a 40% borogluconate solution. In addition, the cows of the treatment group were administered 500 ml of a sodiumphosphate solution intravenously as well as calcium lactate and sodium phosphate orally. Blood samples for the determination of calcium, inorganic phosphorus and magnesium then were collected and the success of the treatment was monitored. Independently of the treatment the mean concentration of calcium transiently increased strongly after the therapy, and then slowly declined again. The phosphorus values of the control cows treated with a calcium infusion alone only rose little while the values of the cows additionally treated with sodium phosphate strongly increased. There was no significant difference in the success rate of treatment in the two groups. The rate of recurrence however was significantly higher in the treatment group than in the control group

Clinical evaluation of the new coat colour macchiato in a male Franches-Montagnes horse

In April 2008 a Franches-Montagnes colt was born with an unusual coat colour phenotype which had never been observed in that population before. The foal showed extended white markings on body and legs, a white head and blue eyes. As both parents have an unremarkable bay coat colour phenotype, a de novo mutation was expected in the offspring and a candidate gene approach revealed a spontaneous mutation in the microphthalmia associated transcription factor gene (MITF). A detailed clinical examination in 2010 indicated an impaired hearing capacity. As in the American Paint Horse large white facial markings in combination with blue eyes are associated with deafness, the hearing capacity of the stallion was closer examined performing brainstem auditory-evoked responses (BAER). The BAER confirmed bilateral deafness in the Franches-Montagnes colt. It is assumed that the deafness is caused by a melanocyte deficiency caused by the MITF gene mutation. Unfortunately, due to castration of the horse, the causal association between the mutation in the MITF gene and clinical findings cannot be confirmed by experimental matings

Proximal melanocyte-specific promoter of the <i>MITF</i> gene.

<p>The depicted region corresponds to positions −89 to +1 with respect to the transcription start site and is in reverse complementary orientation to the genomic reference sequence (ECA16:20,117,350–20,117,261; EquCab2.0). The three vertical red lines in the dog and mouse sequences represent small insertions. In some splashed white horses, an 11 bp motif shown in red replaces an adenine, which is part of a highly conserved PAX3 transcription factor binding site in the wild-type sequence. The inserted 11 bp sequence may have arisen by duplication from an identical sequence motif a few nucleotides further downstream (underlined).</p

Functional validation of the MITF:p.N310S mutation.

<p>(A) The basic DNA binding domain in the vicinity of the mutation is 100% identical in vertebrates from mammals to fish. (B) Electrophoretic mobility shift assay (EMSA). Increasing concentrations (given in µM) of recombinant wild-type and mutant MITF protein from <i>E. coli</i> were incubated with a radioactively labeled double-stranded oligonucleotide and run on a non-denaturing polyacrylamide gel. The mutant MITF protein shows a weaker retention band than the wild-type MITF, which indicates a partially defective DNA binding activity (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002653#pgen.1002653.s008" target="_blank">Table S3</a>).</p

Association of white spotting genotypes in different breeds.

<p>Results for the three newly described splashed white mutations and the overo mutation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002653#pgen.1002653-Metallinos1" target="_blank">[15]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002653#pgen.1002653-Yang1" target="_blank">[17]</a> are shown.</p>a<p>QH, Quarter Horse; AP, American Paint Horse; HN, Hanoverian; IS, Icelandic Horse; MH, Miniature Horse; OL, Oldenburger; SP, Shetland Pony; TB, Thoroughbred; TR, Trakehner; AS, American Standardbred; FM, Franches-Montagnes; HF, Haflinger; NO, Noriker; WB, European Warmblood.</p>b<p>Gene flow exists between QH and AP. Several horses in our study had double registrations with both the American Quarter Horse Association (AQHA) and the American Paint Horse Association (APHA).</p>c<p>The Icelandic Horses with the splashed white or other white phenotype were not experimentally tested for the absence of the <i>EDNRB^I118K</i>, <i>MITF^C280fs*20</i>, and <i>PAX3^C70Y</i> alleles. They were assumed to be homozygous wildtype at these positions.</p

Causative mutations for white spotting phenotypes.

a<p>numbering refers to NM_001163874.1 (<i>MITF</i>) and XM_001494972.3 (<i>PAX3</i>).</p>b<p>build EquCab 2.0.</p

Macchiato coat color phenotype in a Franches-Montagnes stallion.

<p>Note the pronounced depigmentation on the body in addition to the large white blaze on the head, the white legs, and the blue eyes. This horse has a bay basic coat color.</p

Phenotypes of horses with different combinations of splashed white alleles.

<p>(A) American Paint Horse with a very pronounced depigmentation phenotype. In addition to being homozygous for the <i>MITF^prom1</i> allele, it also carries a private allele at the <i>KIT</i> gene (p.H40Q), which may enhance the depigmentation phenotype. The functional significance of the p.H40Q variant is unclear at the moment. (B) This Quarter Horse is also homozygous for the <i>MITF^prom1</i> allele, but has substantially more residual pigmentation than the horse shown in panel A. (C) A Trakehner horse homozygous for the <i>MITF^prom1</i> allele. (D) A completely white horse with multiple splashed white alleles. (E) Quarter Horse with the rare <i>MITF^C280Sfs*20</i> allele. This horse has a very pronounced splashed white phenotype with blue eyes and a largely unpigmented head and belly. (F) A compound heterozygote for two different <i>MITF</i> mutant alleles is completely white. None of the horses in this figure carry the <i>EDNRB^I118K</i> (overo) allele.</p