052-056-WithASplash Modified: December 27, 2019 7:32 AM Files:Publications:PHJ:01-02_20 Journal:01-02_20 Sent To Rip:052-056-WithASplash.indd With a Splash The discovery of Splashed White 5 made waves in 2019—here’s what you need to know.

By JESSICA HEIN

Penny Kleinschmidt helped spark research into Splashed White 5. She owns two Splashed White 5

horses, including TK Ollies After Shock. PENNY KLEINSCHMIDT COURTESY 052-056-WithASplash Modified: December 27, 2019 8:33 AM Files:Publications:PHJ:01-02_20 Journal:01-02_20 Sent To Rip:052-056-WithASplash.indd 052-056-WithASplash Modified: December 27, 2019 8:33 AM Files:Publications:PHJ:01-02_20 Journal:01-02_20 Sent To Rip:052-056-WithASplash.indd

hen APHA member Penny Klein- “Sharing observations and capabilities is schmidt of Indianapolis, Indiana, first looked over her horses’ DNA results, a tremendous way to accelerate science. she was puzzled. The test results indicated her Paints had none of the known alleles capable of The horse owners’ input is really key.” producing white markings. Yet, her horses— TK Ollies After Shock and TK Scotchs After- “After about three decades of molecular shock—weren’t lacking chrome on their coats. genetic research, it has become quite clear Chelsea Dancin Wood was part of the original Meanwhile, across the Atlantic in France, that a small number of genes exist that might research into Splashed another Paint Horse owner had the same ques- cause white-spotting phenotypes in mammals. White 5, along with tion and the same perplexing DNA results. The two most important of the genes are KIT several siblings and her Angelique Levert’s mare, Chelsea Dancin and MITF. There are a few others, but the total sire. Researchers identi- Wood, had plenty of chrome—a bold, mask-like number is less than 10,” Tosso explained; fied a deleted piece of apron face paired with blue eyes, tall stockings “phenotype” refers to the horse’s outward DNA code on the MITF and a swath of white cradling her abdomen and appearance. gene in some horses sharing bold white barrel—and she was also deaf. Surely a genetic KIT (Receptor Tyrosine Kinase) and MITF markings, blue eyes answer was at the core of these mysteries. (Melanocyte Inducing Transcription Factor) and, sometimes, deaf- Both women—separately, but seren- are common culprits for white-spotting pat- ness. This led to the dipitously—reached out to Christa Lafayette, terns. KIT is home to Sabino 1 and the so-called discovery of the founder and CEO of genetic testing company “White” mutations (like W5, W10 and W20), Splashed White 5 allele. Etalon Diagnostics. Intrigued by the loudly marked horses and their corresponding lack of known white-producing alleles, Christa set the wheels in motion to discover a new spotting pattern that was lurking just out of reach of modern genetic testing. Soon, Splashed White 5 was officially discovered. On the Hunt Shortly after the curious owners contacted her in early 2018, Christa jumped on the case from Etalon’s base in Menlo Park, California— she, too, was interested in the potential genetic cause of these boldly marked, bald-faced, blue- eyed beauties. “In the case of Ms. Levert’s mare, we checked our most recent samples for related horses that appeared negative for known white-spotting genetics but displayed obvi- ous white markings and, in this case, blue eyes as well,” Christa explained. “We reached out to the owners of her mare’s siblings, ask- ing detailed questions and requesting more photos. Then, we began second-level testing where we looked in certain ‘suspect’ regions of the horses’ genomes through DNA sequencing to see if we could find anything unusual. The results started to look promising.” Armed with her preliminary suspicions and a battery of sample cases, Christa reached out to genetics researcher Tosso Leeb, Ph.D., at the University of Bern in Switzerland. Intrigued, Tosso agreed to analyze the samples as part of

a larger genome-sequencing project. ANGELIQUE LEVERT COURTESY

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and is closely associated with , as well. cases, deafness. Other sampled horses, who MITF is already home to Splashed White 1 were related by pedigree but did not display and 3. Other genes of interest for Paint breed- similar coat color markings or deafness, were ers include PAX3 (Paired Box 3 Transcription not missing the implicated portion of the MITF Factor), home to Splashed White 2 and 4, and gene. MITF produces a protein that has a key EDNRB (Endothelin Receptor Type B), which is role in the development and function of mela- home to Frame . nocytes, which are pigment-producing cells. “When we learn of a horse with an unex- As such, it can affect skin, hair and eye color, plained white-spotting phenotype, we determine and contributes to the survival of pigment its entire genome sequence, which consists of cells in the inner ear, thereby influencing roughly 2.5 billion nucleotides. Subsequently, hearing function. we focus on the known candidate genes for “The genome of these horses was missing white-spotting phenotypes—which make up a piece of information found on the standard or less than 1 million nucleotides—and analyze ‘wild type’ equine genome,” Christa explained, whether we find any DNA variation that is likely adding that the effect of a genetic deletion can to impact the normal function of these genes,” be minor or major, depending on the extent Tosso explained. of missing information. “We found this same Upon analysis of DNA samples from Chel- deletion in all of the client horses displaying the sea Dancin Wood, her sire Razor N Jills Paco, characteristic bald face, blue eyes, other white and seven additional offspring of that sire, body markings—usually, on the legs—and high Tosso’s team identified a “deletion” of genetic incidence of deafness. Siblings of the loudly Splashed White 5 has code—a section of DNA where some informa- marked horses who did not display white mark- been found in Paints of varied lineage and tion is missing, when compared to the horse ings, blue eyes or deafness did not have the geographical regions. reference genome used by researchers—in deletion we now call Splashed White 5.” Testing is now avail- the MITF gene. This missing section was After discovery of the Splashed White 5 able from APHA’s present in each sample horse that displayed deletion, Christa re-evaluated her other suspect partner labs. bold white markings, blue eyes and, in some horses’ samples and expanded the testing pool.

In the Know: Splashed White 5 Discovery: 2019, University of Bern (Switzerland) Location: MITF gene on chromosome 16 Note: This is the same gene affected by Splashed White 1 and Splashed White 3 Cause: A deleted section of the MITF gene, which is required for the survival of pigment- producing cells Distribution: Confirmed in a number of different pedigrees and geographic loca- tions; suspected to go to at least the 1970s, per Etalon researchers, though more samples will help narrow down a suspected founding horse. Characteristics: Bald face, blue eyes, and white leg/abdomen markings are common; associated deafness in some horses Testing: Available as a special request through Etalon Diagnostics; available at the University of California–Davis as part of the Splashed White test and APHA Color/Pattern panel (beginning December 2019) The Science: Search “white spotted horse” on ncbi.nlm.nih.gov/pubmed to read the

COURTESY AMY COURTESY CHANDLER research abstract 052-056-WithASplash Modified: December 26, 2019 11:22 AM Files:Publications:PHJ:01-02_20 Journal:01-02_20 Sent To Rip:052-056-WithASplash.indd 052-056-WithASplash Modified: December 26, 2019 11:22 AM Files:Publications:PHJ:01-02_20 Journal:01-02_20 Sent To Rip:052-056-WithASplash.indd

Sure enough, they also had the MITF deletion; Vocab Lesson Penny’s horses, TK Ollies After Shock and Allele—variants of a given gene TK Scotchs Aftershock, as well as ALC Hope Genotype—an organism’s genetic makeup Always Wins, a 2010 overo mare owned Phenotype—an organism’s outward appearance by Amy Chandler of Morriston, Florida, joined Genome—an organism’s complete genetic makeup the list of confirmed Splashed White 5 horses. Deletion—a section of genetic code that’s missing from the area where “What’s truly amazing about this kind it’s typically located in a species of collaboration is that the horse owners Mutation—a deviation from the standard genetic makeup of a species get their answers; new genetic tools are MITF—Melanocyte Inducing Transcription Factor; this gene produces a discovered and made available to others; the protein that affects the development and function of pigment-producing academic scientists achieve a publication in melanocytes record time; and APHA gets a new pattern. Increase your Paint genetics know-how by reading these past Paint Horse Everybody wins,” Christa said. “Sharing Journal articles: observations and capabilities is a tremendous • “ The Color Inside—Parts 1 & 2:” January 2016 and February 2016 way to accelerate science. The horse owners’ Read this article free online at apha.com/breed/geneticeq input is really key here; without their involve- • “In the Cards:” February 2017 • “Reading Between the Lines:” September/October 2018 ment, photos and observations, we would not have had any clues as to what was lying undiscovered in the DNA samples.” Puzzle Pieces Splashed White 5’s discovery on Though research into Splashed White 5 is new, the following characteristics seem to be associ- MITF reinforces the fact that almost all ated with this pattern: white-spotting alleles are concentrated • Bald face • Blue eye(s) in a few specific gene locations. • White lower legs, with markings sometimes extending on the barrel/abdomen (giving a “dipped in paint” look) cannot be solely determined by looking at the • A ssociated deafness in some, but not all, phenotype of a horse and/or the genotypes of horses the parents, so breeders need to know whether a “The increased risk for deafness is horse carries SW5 or not.” directly caused by the MITF variant,” Tosso Both APHA partner laboratories—Etalon explained. “The inner ear needs some pig- Diagnostics and University of California– ment cells to maintain its function. If the Davis—offer testing for Splashed White 5. At inner ear is completely devoid of pigment Etalon, it’s an a la carte option available for $40 cells, it will degenerate and become deaf. and it’s not included on the All-Inclusive Mini- Therefore, any genetic variants that lead to Panel for APHA at this time. Through UC–Davis, white heads are likely to increase the risk for the test is included on the APHA Color & Pattern deafness. MITF variants are known to also Panel and also offered as part of the individual cause deafness in humans. Splashed White test, as of December 2019. “Based on knowledge from other spe- Ultimately, the official identification of cies, SW5 is likely to act as a semi-dominant Splashed White 5 has several direct implications allele,” Tosso continued; this means inheriting for Paint Horse breeders. one copy of Splashed White 5 has a different First, discovery of Splashed White 5 provides effect on the horse than when two copies are Paint owners with greater insight—a genetic inherited. “One copy of SW5 produces a white- “answer” as to the cause of some horses’ dis- spotting phenotype and an increased risk for tinctive markings and, in some, potential for deafness. Two copies of SW5 in a homozygous hearing impairment. Next, it offers breeders a horse will completely abolish MITF function new tool—either in color production potential and likely result in a very severe phenotype, or as a means to avoid the associated compli- causing blindness, deafness, completely white cations. Third, Splashed White 5’s discovery body color, potentially reduced lifespan or peri- on MITF, a gene that’s well-established for natal lethality. its white-spotting mutations, reinforces the “There are many different white-spotting fact that almost all white-spotting alleles are phenotypes in horses. Often the exact genotype concentrated in a few specific gene locations in

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horses—so while Mother Nature might shake Penny said about her 2017 sorrel overo stallion; things up once in a while with a new mutation, “Chuck” made his showing debut in 2018 with the genes at the root of most white markings are Penny in in-hand events, and they progressed to well defined. riding events in 2019. “I intend to continue work- ing on building his skills in different disciplines The Road Ahead to eventually make him an awesome all-around For Penny, discovery of Splashed White 5 horse. Chuck has the biggest heart and always doesn’t change the plans she has for her Paints, works hard to please, no matter what’s asked of but it’s gratifying to now have an answer to him. It’s also thrilling and inspiring to be part of what once was a genetic mystery. a new color gene’s discovery.” “I am the proud breeder and owner of TK Ollies After Shock—a special connection started Jessica Hein is editor of the Paint Horse Journal. between us as I watched him being born,” [email protected]

Confirmed Splashed White 5 Cases COURTESY AMY COURTESY CHANDLER PENNY KLEINSCHMIDT COURTESY ALC Hope Always Wins TK Ollies After Shock 2010 sorrel overo mare 2017 sorrel overo stallion Intensely Handsome x Sonnys Lucky Image Sonnys After Shock x Ollies New Design Owned by Amy Chandler, Morriston, Florida Owned by Penny Kleinschmidt, Indianapolis, Indiana SW5/n, W20/n SW5/n COURTESY ANGELIQUE ANGELIQUE LEVERT COURTESY THE HORSE PHOTOGRAPHY OF EYE Chelsea Dancin Wood TK Scotchs Aftershock 2016 overo mare 2017 sorrel overo gelding Razor N Jills Paco x Southern Dottie Sonnys After Shock x Annies Classy Scotch (QH) Formerly owned by Angelique Levert, Villeneuve Owned by Penny Kleinschmidt, Indianapolis, Du Latou, France; now owned by Sarah Grand Indiana SW5/n, W20/W20 SW5/n

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