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SHORT COMMUNICATION
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
V. J. Utzeri, F. Bertolini, A. Ribani, G. Schiavo, S. Dall’Olio and L. Fontanesi
Department of Agricultural and Food Sciences, Division of Animal Sciences, University of Bologna, Viale Fanin 46, 40127 Bologna, Italy.
Summary 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=6.17E18). 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.
Keywords Asinara island, coat colour, deleterious mutation, equid, oculocutaneous
albinism Type 1, pigmentation, population genetics
Asinara (of which one of its middle age etymology seems to
recall the meaning of ‘land of the donkeys’) is a small
Mediterranean island (about 52 km
2
) located closely north-
west of Sardinia (Fig. 1a). This island was inhabited until
1885, at which time it was closed as it became an Italian
quarantine site and subsequently a highly secured prisoner
colony. The island was re-opened to the public in 1999 after
the constitution of the Asinara National Park in 1998
(http://www.parcoasinara.org: Gazzetta Ufficiale della
Repubblica Italiana 1997). Among the species living on
the island, the Asinara white donkey (Fig. 1b) or ‘Asino
dell’Asinara’ (Equus asinus) is the most representative and
peculiar component of the Park’s fauna and is the symbol of
Asinara. The origin of the Asinara white donkeys is
uncertain and based only on legends, from which it is
possible to date the occurrence of the first white donkeys
back to before the closure of the island in the 19th century
(Ministero di Agricoltura, Industria e Commercio 1905;
Vinceti 2007). The Asinara white donkey subpopulation or
colour morph (accounting for ~100–120 animals) lives
together with coloured (usually grey) donkeys that can be
attributed to the Asino Sardo population (Pinna et al.
1993). All these donkeys can mate, producing a hybrid
population (whose number of heads is not known). The
whole donkey population of Asinara island (white and
coloured) can be considered a feral population, as no
human direct intervention has been managing these
animals for more than a century (Kugler & Broxham
2014). The white coat coloured animals are also considered
by the Food and Agriculture Organization (Sherf 2000) and
by the register of equine and asinine Italian local breeds
Address for correspondence
L. Fontanesi, Department of Agricultural and Food Sciences, Division of
Animal Sciences, University of Bologna, Viale Fanin 46, 40127 Bologna,
Italy.
E-mail: luca.fontanesi@unibo.it
Accepted for publication 12 October 2015
doi: 10.1111/age.12386
1
©2015 Stichting International Foundation for Animal Genetics
(Ministero delle Politiche Agricole, Alimentari e Forestali
2010) as a donkey breed in critical status.
Despite the uniqueness of the Asinara white donkeys,
only a few authors have investigated this subpopulation. As
far as we know, these animals have been analysed at the
DNA level using microsatellites to evaluate genetic
variability together with other donkey breeds in only two
studies (Cosseddu et al. 2001; Colli et al. 2013). Pinna et al.
(1993) described the Asinara white donkeys at the
morphological level and reported that these animals
resemble those of the Asino Sardo breed in terms of size
and body shape, confirming their genetic closeness deter-
mined by microsatellite markers (Colli et al. 2013). The
differentiating trait is only the complete white coat colour,
lacking pigmentation in the skin, hair, eyelashes and
eyebrows, and eyes that are light blue, as also described
for several forms of human oculocutaneous albinism Type
1A and 1B (OCA1A and OCA1B) defects (e.g. Grønskov
et al. 2007; Fig. 1c). These donkeys have low visual acuity,
and during sunny hours they hide inside the unused
buildings of the prisoner colony. These traits and their
evasive behaviour away from the sun indicate that Asinara
white donkeys are affected by albinism (Pinna et al. 1993).
The albinism in these animals is one of the few cases of this
type of pigmentation defect that is maintained in a wild or
feral vertebrate population (Protas et al. 2006; Xu et al.
2013), as fitness is expected to be lower, especially in a
sunny Mediterranean environment.
In many different species, the albino locus allelic series
(formally identified as the Clocus; Searle 1968) is
determined by mutations in the tyrosinase (TYR) gene that
lead to completely white coat colour and lack of pigmen-
tation in the case of disrupting mutations (Aigner et al.
2000; Oetting 2000; Beermann et al. 2004; Schmutz et al.
2004; Blaszczyk et al. 2005; Imes et al. 2006; Blaszczyk
et al. 2007; Anistoroaei et al. 2008), determining the
recessive callele(s) (Searle 1968). Tyrosinase (EC
1.14.18.1) is the key enzyme involved in the melanogenesis
process in which both melanins (eumelanims and pheome-
lanins) are produced. This enzyme has an active site
(a) (b) (c)
(d) (e)
Figure 1 Geographical position of the Asinara island, phenotypic details of white Asinara donkeys and the causative mutation determining their
albinism. (a) Geographical location of the Asinara island. (b) Asinara white albino donkey. (c) A close-up of the depigmented eye of an Asinara white
albino donkey. (d) Recessive Mendelian inheritance of the albino phenotype demonstrated in a trio. Two grey parents (with heterozygous genotype
H/D at the p.His202Asp site or C/G in the nucleotide sequence at the c.604C>G nucleotide position, indicated with S, according to the IUPAC
nomenclature) gave birth to an albino donkey (D/D genotype or G/G at the nucleotide position). Microsatellite analysis (data not shown) confirmed
the relationship among the three donkeys. (e) Alignment of the donkey tyrosinase protein region containing the p.His202Asp substitution with the
corresponding region in different species. The grey region indicated with an arrow corresponds to the position of the p.His202Asp substitution in
donkeys (H2A position in the CuA site). The other arrow indicates the histidine of the H3A position in the CuA site (the H1A position is not included in
this alignment). Protein accession numbers for the sequences used in the alignment are as follows: Equus caballus, F6YIA2; Homo sapiens, P14679;
Mus musculus, P11344; Bos taurus, Q8MIU0; Oryctolagus cuniculus, G1SYA0; Gallus gallus, P55024; Xenopus laevis, F7CL37; Danio rerio,
F1QDZ4; Ipomoea batatas, Q9MB14; Neurospora crassa, P00440; Bacillus megaterium, B2ZB02. Numbers in the alignments indicate the starting
and ending amino acid residues of the corresponding protein.
©2015 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12386
Utzeri et al.2
composed of a pair of antiferromagnetically coupled copper
ions, CuA and CuB, which are coordinated by six histidine
residues, three per each copper-binding site (Claus & Decker
2006; Kanteev et al. 2015). Removal of only one of the
copper-binding histidine residues results in loss of the
corresponding copper ion, thereby abolishing enzyme
activity (e.g. Jackman et al. 1991).
In this study, we used a candidate gene approach to
identify the causative mutation of the albinism in Asinara
white donkeys. For this aim, six primer pairs (Table S1)
were designed on the assembled donkey TYR gene
(Bertolini et al. 2015) and used to amplify and sequence
by Sanger and Ion Torrent sequencing technologies (as
described in Fontanesi et al. 2015) all coding exons,
portions of the intronic regions (downstream and
upstream of the exons), 50- and 30- untranslated regions
of the donkey TYR gene in 13 animals of different coat
colours (seven Asinara white donkeys, expected to have
the c/c genotype at the albino locus, and six coloured
donkeys: two grey Asinara donkeys, phenotypically con-
sidered as Asino Sardo donkeys; one Asino Sardo donkey;
one Martina Franca; one Sicilian Grey; and one Ragusano;
EMBL accession numbers LN880531 and LN880532).
Seven single nucleotide polymorphisms (SNPs) were iden-
tified (Table S2). Four SNPs were in exonic regions (three
in exon 1 and one in exon 2), and the remaining
polymorphisms were in intronic regions (two in intron 2
and one in intron 4; Table S2). Of the four missense
mutations, two (c.274G>A or p.Val83Ile in exon 1 and
c.987G>A or p.Glu316Lys in exon 2) were identified only
in the heterozygous condition in one coloured donkey
(Ragusano). The SIFT score (Kumar et al. 2009) indicated
that these two amino acid substitutions are tolerated
(Table S2). For the c.18G>C or p.Leu6Phe mutation, the
genotype for three coloured donkeys of different breeds
(Martina Franca, Grigio Siciliano and Ragusano) was G/G
(L/L), whereas it was heterozygous G/C (L/F) in two grey
donkeys sampled in the Asinara island (resembling Asino
Sardo donkeys) and homozygous C/C (F/F) in the third
grey Asino Sardo donkey sampled in Sardinia. Genotype
C/C or F/F was fixed in all Asinara white albino donkeys
as well (Table S2). SIFT analysis indicated that this
missense mutation is not deleterious (P=0.48). The
second missense mutation (c.604C>G or p.His202Asp;
Fig. 1d), identified only in donkeys from Asinara island
that were homozygous D/D in all sequenced white
donkeys, had a highly significant SIFT score (P<0.001)
supporting the deleterious effect of this substitution
(Table S2).
The amino acid at position 202 of the wild-type TYR
protein is one of the three highly conserved histidine
positions of the first copper-binding site (CuA) of the TYR
catalytic domain (Fig. 1e). This histidine is the second
copper-binding histidine residue within the CuA site (indi-
cated as H2A) that is always present at this position in all
tyrosinase protein sequences available, even across king-
doms (Fig. 1e; Garc
ıa-Borr
on & Solano 2002; Claus &
Decker 2006). The 3D structure of the wild-type and
mutated donkey TYR proteins obtained following the
homology modelling strategy (template protein: PDB entry
4P6R of Bacillus megaterium; Goldfeder et al. 2014) with
MODELLER software (version 9.14; Eswar et al. 2006) con-
firmed the disruptive effect of the p.His202Asp substitution
(Fig. S1).
According to the sequencing data, as grey donkeys
sampled on the Asinara island were heterozygous at the
c.604C>G (p.His202Asp) missense mutation, it was possible
to presume a recessive mode of inheritance of the effect of
the mutated allele, as expected for mutations causing
albinism (Searle 1968). Mendelian recessive inheritance of
this mutation was strengthened by sequencing and
genotyping (Table S1) a trio family sampled on the Asinara
island composed of a grey father (genotype C/G or H/D), a
grey mother (C/G or H/D) and a white albino foal (G/G or D/
D; Fig. 1d).
To further confirm the role of the p.His202Asp substitu-
tion, the c.604C>G mutation was genotyped (Table S1) in a
total of 65 donkeys (including the animals already
sequenced to confirm the sequencing determined genotype)
from eight coloured breeds or populations in addition to 17
feral Asinara white albino donkeys (Table 1). All Asinara
white albino donkeys were homozygous for the mutated
allele. Only four grey Asino Sardo donkeys were heterozy-
gous (three sampled in Asinara National Park, of which the
sequencing of two have already been described and
the third being from a farm in the province of Sassari, in
the north of Sardinia). Considering all genotyped donkeys of
Table 1 Distribution of c.604C>G (p.His202Asp) genotypes obtained
from PCR-RFLP and sequencing analyses among the investigated
breeds.
Donkey breeds/populations No. of donkeys
c.604C>G genotypes
1
C/C C/G G/G
Amiata (coloured) 2 2 ––
Asinara (white albino) 17 –– 17
Asino Sardo (coloured)
2
734–
Coloured hybrids (coloured) 13 13 ––
Martina Franca (coloured) 13 13 ––
Pantesco (coloured) 1 1 ––
Ragusano (coloured) 19 19 ––
Romagnolo (coloured) 2 2 ––
Sicilian Grey (coloured) 8 8 ––
Total 82 61 4 17
1
Including also grey donkeys sampled in Asinara island (see text for
details).
2
The number of donkeys with the corresponding genotype is reported.
Genotypes are indicated for the c.604C>G single nucleotide
polymorphism: allele C corresponds to the deduced amino acid histidine
and allele G corresponds to the deduced amino acid aspartic acid for the
missense mutation indicated as protein position (p.His202Asp).
©2015 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12386
TYR gene mutation and albinism in donkey 3
different breeds and populations and the occurrence of
homozygous G/G (D/D) animals only in albino donkeys,
across-population association between the genotype at the
c.604C>G mutation and the albino phenotype was highly
significant (P=6.17E18; two-tailed chi-squared test).
The phylogenetic tree produced including the five donkey
TYR haplotypes (obtained from the sequenced donkeys using
PHASE program v. 2.1; Stephens et al. 2001) and the horse
sequence (Wade et al. 2009), generated with the UPGMA
method available in MEGA6 software (Tamura et al. 2013),
supported the hypothesis that the albino mutation occurred
in a ‘grey’ haplotype also present in Sardinia donkeys
(Fig. S2). This hypothesis might exclude the legendary origin
of the white donkeys of Asinara that had them deriving from
white Egyptian donkeys imported by Marchese di Mores,
Duke of Asinara Island, in the 19th century, or from a
French shipwreck in the same period (Vinceti 2007).
The isolation of the Asinara donkey population and the
consequent putative high inbreeding level might have been
the causes of the increased frequency of the TYR-mutated
allele in the Asinara island donkey population. The presence
of many small uninhabited tumbledown buildings left over
from previous uses of the island that are used as shelters by
the white donkeys during the sunniest period of the year
and the low activity of these animals during daylight might
reduce the negative effects of this mutation. However, we
cannot be sure whether these hypotheses are sufficient to
explain the conservation of a mutation determining a
potential deleterious effect in a free-living population (Page-
McCaw et al. 2004). We did not investigate whether the
albino TYR haplotype is in linkage disequilibrium with other
variant(s) conferring advantages in a wild, marginal and
harsh environment.
The identification of the causative mutation of the
albinism in the Asinara white donkeys adds a new natural
animal model for human OCA1 defects and might open new
perspectives to study the dynamics of this putative delete-
rious allele in a feral population and to manage this
interesting animal genetic resource that is the symbol of the
Asinara National Park.
Acknowledgements
We thank Giovannatonio Pilo (Asinara National Park
veterinarian), Dr. Guy D’Hallewin (CNR Sassari), Marco
Ghionda (ANAS), Asineria of Gombola, Asinara National
Park personnel for their help during the sampling and
Samuele Bovo and Paolo Manghi (University of Bologna) who
helped with the 3D modelling. This work has been possible
with the collaboration of the Asinara National Park and with
the RFO2014 funds provided by the University of Bologna.
Conflict of interest
The authors declare they have no conflict of interests.
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Supporting information
Additional supporting information may be found in the
online version of this article.
Figure S1 3D modelled structure of the (a) wild type (allele
H–His at position 202) and (b) mutated (allele D –Asp at
position 202) TYR proteins in the CuA and CuB copper-
binding sites.
Figure S2 Phylogenetic tree of the donkey TYR gene
haplotypes.
Table S1 PCR primers used in this study, sequencing and
genotyping.
Table S2 TYR gene polymorphisms and genotypes of the
sequenced donkeys.
©2015 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12386
TYR gene mutation and albinism in donkey 5