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Whole-genome sequencing reveals a large deletion in the MITF gene in horses with white spotted coat colour and increased risk of deafness

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White spotting phenotypes in horses are highly valued in some breeds. They are quite variable and may range from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for white spotting phenotypes in horses. For the present study, we investigated an American Paint Horse family segregating a phenotype involving white spotting and blue eyes. Six of eight horses with the white‐spotting phenotype were deaf. We obtained whole‐genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~63‐kb deletion spanning exons 6–9 of the MITF gene (chr16:21 503 211–21 566 617). We confirmed the breakpoints of the deletion by PCR and Sanger sequencing. PCR‐based genotyping revealed that all eight available affected horses from the family carried the deletion. The finding of an MITF variant fits well with the syndromic phenotype involving both depigmentation and an increased risk for deafness and corresponds to human Waardenburg syndrome type 2A. Our findings will enable more precise genetic testing for depigmentation phenotypes in horses.
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SHORT COMMUNICATION
Whole-genome sequencing reveals a large deletion in the MITF
gene in horses with white spotted coat colour and increased risk of
deafness
J. Henkel*
, C. Lafayette
, S. A. Brooks
§
, K. Martin
, L. Patterson-Rosa
§
, D. Cook
,
V. Jagannathan*
and T. Leeb*
*Institute of Genetics, Vetsuisse Faculty, University of Bern, 3001, Bern, Switzerland.
DermFocus, University of Bern, 3001, Bern,
Switzerland.
Etalon Inc., Menlo Park, CA 94025, USA.
§
Department of Animal Sciences, University of Florida, Gainesville, FL 32611-0910,
USA.
Summary White spotting phenotypes in horses are highly valued in some breeds. They are quite
variable and may range from the common white markings up to completely white horses.
EDNRB,KIT,MITF,PAX3 and TRPM1 represent known candidate genes for white spotting
phenotypes in horses. For the present study, we investigated an American Paint Horse
family segregating a phenotype involving white spotting and blue eyes. Six of eight horses
with the white-spotting phenotype were deaf. We obtained whole-genome sequence data
from an affected horse and specifically searched for structural variants in the known
candidate genes. This analysis revealed a heterozygous ~63-kb deletion spanning exons 69
of the MITF gene (chr16:21 503 21121 566 617). We confirmed the breakpoints of the
deletion by PCR and Sanger sequencing. PCR-based genotyping revealed that all eight
available affected horses from the family carried the deletion. The finding of an MITF
variant fits well with the syndromic phenotype involving both depigmentation and an
increased risk for deafness and corresponds to human Waardenburg syndrome type 2A. Our
findings will enable more precise genetic testing for depigmentation phenotypes in horses.
Keywords Equus caballus, heterogeneity, leucism, melanocyte, pigmentation, splashed
white, structural variant
White spotting in horses and other mammals may result
from an altered embryonic development of the neural crest
melanocyte lineage and a lack of mature melanocytes in the
unpigmented skin areas (‘leucism’). Candidate genes for
such phenotypes in the horse include EDNRB,KIT,MITF,
PAX3 and TRPM1 (Thomas & Erickson 2008; OMIA
000629, 000209, 001688, 000214, 001341).
The horse currently represents the species with the
largest number of molecularly defined white spotting alleles
among domesticated animals. A missense variant in the
EDNRB gene causes the frame overo white spotting pattern
or lethal white foal syndrome if present in heterozygous or
homozygous state respectively (Santschi et al. 1998). Vari-
ants in MITF and PAX3 cause the so-called splashed white
phenotype, which closely resembles human Waardenburg
syndrome (Pingault et al. 2010; Hauswirth et al. 2012,
2013; D
urig et al. 2017). A variant in the equine TRPM1
gene causes the so-called leopard complex spotting (Bellone
et al. 2013). Furthermore, according to our knowledge, 29
different functional alleles at the equine KIT gene have been
described so far. These include the alleles for sabino-1 and
tobiano spotting as well as an allelic series termed W1W27
(Brooks & Bailey 2005; Brooks et al. 2007; Haase et al.
2007, 2008, 2009, 2011, 2015; Holl et al. 2010;
Hauswirth et al. 2013; Capomaccio et al. 2017; Hoban
et al. 2018; Table S1).
We studied a family of American Paint horses segregating
for a white spotting phenotype resembling the splashed
white pattern that could not be explained by any of the
previously published white spotting alleles (Fig. 1). We had
access to samples from one affected stallion and eight of his
daughters (Fig. S1). Seven of the daughters also had white
spotting patterns that could not be explained by their
genotypes at the known white spotting variants. One of the
daughters had a regular frame overo phenotype, which was
Address for correspondence
T. Leeb, Institute of Genetics, Vetsuisse Faculty, University of Bern,
Bremgartenstrasse 109a, 3001 Bern, Switzerland.
E-mail: tosso.leeb@vetsuisse.unibe.ch
Accepted for publication 12 December 2018
doi: 10.1111/age.12762
1
©2019 Stichting International Foundation for Animal Genetics
inherited from the dam. The coat colour phenotypes in this
family were quite variable, as all horses carried additional
known white-spotting alleles. All affected horses had white
faces. The amount of body pigmentation was variable and
one of the studied horses was even completely white. At
least six of the affected horses had blue eyes. The quality of
our photos of the two remaining affected horses did not
allow unambiguous determination of their eye colour. Six
out of eight horses with unexplained white spotting
phenotypes were deaf.
We prepared an Illumina TruSeq PCR free genomic DNA
library and re-sequenced the genome of one affected horse
at 199coverage using 2 9150 bp reads on an Illumina
NovaSeq 6000 instrument. Sequencing and read mapping
to the EquCab 3 reference assembly was performed as
previously described (Jagannathan et al. 2018). Data were
deposited at the European Nucleotide Archive (study
accession no. PRJEB14779, sample SAMEA4822838). This
analysis failed to reveal any single nucleotide or small indel
variants in the candidate genes EDNRB,KIT,MITF, PAX3
and TRPM1. To search for large structural variants, we
visually inspected the read alignments for these genes in the
INTEGRATIVE GENOMICS VIEWER (IGV; Robinson et al. 2011).
The analysis revealed a large heterozygous deletion
spanning the last four exons of the MITF gene correspond-
ing to exons 69 of the MITF-M transcript isoform (Fig. 2).
Specifically, 63 407 bp were missing (chr16:21 503 211
21 566 617). There is currently no RefSeq entry for the
equine melanocyte-specific MITF-M transcript isoform
available. We therefore used the accessions JN896378.1
(mRNA) and AFH66983.1 (protein) to analyse the putative
effects of the deletion on the transcript and protein.
Assuming regular splicing and polyadenylation of the
remaining exons, the deletion removes 706 nucleotides
(56%) from the open reading frame (JN896378.1:
c.555_1260del). This includes the codons required to
encode the functionally important bHLH-Zip domain
required for DNA binding. We did not find any other
obvious structural variants in the EDNRB,KIT, PAX3 and
TRPM1 genes.
We designed primers flanking the deletion (TTAGCAA
TAAGCCACTGGTC, TCATTGTGTCCAGGCTGCTG) and con-
firmed the breakpoints of the deletion by PCR and Sanger
sequencing (Fig. 2). A PCR with these primers and ATG360
polymerase (ThermoFisher) was used as diagnostic assay to
genotype additional horses for the deletion. All eight
affected horses from this family, from which genomic DNA
was available, carried the deletion. The phenotype resem-
bling the splashed white pattern with an extremely large
blaze on the head and frequently associated with blue eyes
is similar to the phenotypes of horses with other mutant
MITF alleles. Most of the horses carrying this deletion were
(a) (b)
(c) (d) Figure 1 White spotting phenotype resem-
bling the splashed white pattern. (a, b) This
horse had light blue eyes, a white face, white
legs and a small white belly spot. The horse
was deaf. (c, d) This horse had a more
pronounced white spotting phenotype com-
pared to its full sibling shown in (a) and (b). It
also had light blue eyes and was deaf. Geno-
types at important white spotting loci are
indicated. MITF
del
designates the new MITF
allele identified in this study.
Figure 2 Details of the MITF deletion. (a) A coverage plot of the whole
genome sequence data indicates a heterozygous ~63-kb deletion
comprising exons 69 of the MITF gene. (b) Sanger sequencing of a PCR
product from the deletion allele precisely defined the breakpoints of the
deletion (Chr16:21 503 21121 566 617del, EquCab 3 assembly).
©2019 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12762
Henkel et al.2
deaf. This confirms earlier observations that horses with a
lack of functional MITF have an increased risk for deafness
(Hauswirth et al. 2012). All deaf horses carried additional
white spotting alleles, which may have exacerbated the
functional impact of the MITF deletion.
In conclusion, our study revealed a large structural
variant at the equine MITF gene, which most likely causes a
splashed white depigmentation phenotype and predisposes
to deafness.
Acknowledgements
The authors would like to thank all involved horse owners
for donating samples and pictures and for sharing pedigree
information of their horses. The authors also wish to thank
Nathalie Besuchet, Muriel Fragni
ere and Sabrina Schenk for
expert technical assistance. The Next Generation Sequenc-
ing Platform of the University of Bern is acknowledged for
performing the whole genome re-sequencing experiments
and the Interfaculty Bioinformatics Unit of the University of
Bern for providing high performance computing infrastruc-
ture. This study was supported by grant 31003A_172964
from the Swiss National Science Foundation.
Conflicts of interest
Christa Lafayette, Katie Martin and Deborah Cook are
affiliated with a genetic testing laboratory offering tests for
white spotting in horses.
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Supporting information
Additional supporting information may be found online in
the Supporting Information section at the end of the article.
Figure S1 Pedigree of the studied American Paint Horse
family.
Table S1 Variants, horses and their genotypes.
©2019 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12762
Equine MITF deletion 3
... Among domesticated mammals, the horse (Equus caballus) currently possesses the most variants associated with white spotting patterns (Henkel et al., 2019). Forty-three genetic variants, mostly located in the KIT proto-oncogene, receptor tyrosine kinase (KIT) and the melanocyte inducing transcription factor (MITF) genes, are implicated in white spotting and depigmentation phenotypes in the horse (Brooks et al., 2008;Brooks & Bailey, 2005;Dürig et al., 2017;Haase et al., 2007;Haase et al., 2009;Haase et al., 2011;Haase et al., 2015;Hauswirth et al., 2012;Hauswirth et al., 2013;Holl et al., 2010;Holl et al., 2017;Martin et al., 2020;Metallinos et al., 1998;Patterson Rosa et al., 2021). ...
... Phenotypes for white spotting alleles can range from small white markings on the extremities, as observed with KIT W20 , to complete white phenotypes in other mutant MITF or multiallelic individuals. The splashed white (SW) phenotype is associated with variants in two genes, MITF and PAX3, and is phenotypically similar to the human Waardenburg syndrome (Dürig et al., 2017;Hauswirth et al., 2012;Hauswirth et al., 2013;Henkel et al., 2019;Magdesian et al., 2020;Pingault et al., 2010). MITF variants have also been associated to diminished or lacking auditory response in heterozygotes, as in SW5 (Henkel et al., 2019). ...
... The splashed white (SW) phenotype is associated with variants in two genes, MITF and PAX3, and is phenotypically similar to the human Waardenburg syndrome (Dürig et al., 2017;Hauswirth et al., 2012;Hauswirth et al., 2013;Henkel et al., 2019;Magdesian et al., 2020;Pingault et al., 2010). MITF variants have also been associated to diminished or lacking auditory response in heterozygotes, as in SW5 (Henkel et al., 2019). ...
... Table S4: Allele frequencies for each coat color locus analyzed in this study after filtering for relatedness across breeds. References [41,42] are cited in the supplementary materials. ...
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