Reprod Dom Anim 51, 171174 (2016); doi: 10.1111/rda.

12636
ISSN 09366768

Short Communication
A Non-Reciprocal Autosomal Translocation 64,XX, t(4;10)(q21;p15) in an Arabian
Mare with Repeated Early Embryonic Loss
S Ghosh1, PJ Das2, F Avila3, BK Thwaits4, BP Chowdhary5 and T Raudsepp1
1
Texas A&M University, College Station, TX, USA; 2National Research Centre on Yak, Dirang, Arunachal Pradesh, India; 3Department of
Veterinary Population Medicine, University of Minnesota, St. Paul, MN, USA; 4Rogers Bandalero Ranch, Benson, AZ, USA; 5Qatar University,
Doha, Qatar

Contents
Balanced autosomal translocations are a known cause for
repeated early embryonic loss (REEL) in horses. In most cases,
carriers of such translocations are phenotypically normal, but
the chromosomal aberration negatively aects gametogenesis
giving rise to both genetically balanced and unbalanced
gametes. The latter, if involved in fertilization, result in
REEL, whereas gametes with the balanced form of translocation will pass the defect into next generation. Therefore, in
order to reduce the incidence of REEL, identication of
translocation carriers is critical. Here, we report about a
phenotypically normal 3-year-old Arabian mare that had
repeated resorption of conceptuses prior to day 45 of gestation
and was diagnosed with REEL. Conventional and molecular
cytogenetic analyses revealed that the mare had normal
chromosome number 64,XX but carried a non-mosaic and
non-reciprocal autosomal translocation t(4;10)(q21;p15). This
is a novel translocation described in horses with REEL and the
rst such report in Arabians. Previous cases of REEL due to
autosomal translocations have exclusively involved Thoroughbreds. The ndings underscore the importance of routine
cytogenetic screening of breeding animals.

Introduction
Subfertility due to early pregnancy failure before days
4060 of gestation is a recognized economic problem for
the equine industry (Ball 1988; Vanderwall 2008). In
case of repeated early embryonic loss (REEL), the
frequency of pregnancy failure can be as high as 50%
(Ball 1988). Several factors have been associated with
REEL, including the eect of uterine and oviduct
environment, hormonal imbalance, maternal age,
embryonic defects and genetic factors (Ball 1988).
Knowledge about the latter is currently limited to a
handful of cases with balanced chromosomal aberrations (Lear and Bailey 2008).
Balanced translocations result from reciprocal or
non-reciprocal exchange between non-homologous
chromosomes so that, overall, no genetic material is
lost or gained (see Villagomez and Pinton 2008).
Therefore, balanced translocations typically do not
aect the health or viability of the carrier, but they
disturb chromosome pairing and segregation during
gametogenesis, resulting in the formation of both

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genetically balanced and unbalanced gametes. Among

balanced gametes, there are cells with normal chromosomes as well as cells with the balanced form of
translocation, whereas approximately two-thirds of the
gametes are genetically unbalanced (see Durkin et al.
2011). The latter, if involved in fertilization, will cause
REEL, while gametes with a balanced translocation
will result in normal ospring but recreate the chromosomal abnormality and predisposition to REEL in
the next generation.
To date, eight cases of balanced autosomal translocations, six in mares (Power 1991; Lear and Layton
2002; Lear et al. 2008, 2014) and two in stallions (Long
1996; Durkin et al. 2011), have been reported for
horses all described in Thoroughbreds with REEL.
Here, we report the rst such case in an Arabian mare.

Material and Methods

Case description and sampling
A phenotypically normal 3-year-old Arabian mare was
presented for karyotyping after being diagnosed with
REEL. Clinical reports indicated that the mare had
repeated resorption of conceptuses prior to day 45 of
gestation, with or without progesterone administration.
By the time of chromosome analysis, she had had no live
foals. Very recently, however, the mare gave birth to a
live foal, but this was not available for sampling.
Peripheral blood from the mare was collected in Naheparin vacutainers (VACUTAINERTM, Becton Dickinson, Franklin Lakes, NJ, USA) and used for cell
cultures and chromosome preparations.
Chromosome preparations and karyotyping
Metaphase chromosome spreads were prepared from
Pokeweed-stimulated blood lymphocyte cultures
according to standard protocols (Raudsepp and
Chowdhary 2008). Chromosomes were stained with
Giemsa for initial counting; sex chromosomes were
identied by CBG banding (Arrighi and Hsu 1971);
rened chromosome analysis and karyotyping were
carried out by GTG banding (Seabright 1971). Twenty

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S Ghosh, PJ Das, F Avila, BK Thwaits, BP Chowdhary and T Raudsepp

cells were captured and analysed for each technique

using an Axioplan2 microscope (Carl Zeiss, Inc., Jena,
Germany) and IKAROS (MetaSystems GmbH, Altlussheim, Germany) software.
Fluorescence in situ hybridization (FISH)
Identication of aberrant chromosomes and demarcation of translocation breakpoints were carried out by
FISH to metaphase/interphase chromosomes using

horse bacterial articial chromosome (BAC) clones

(CHORI-241: http://bacpac.chori.org/equine241.htm)
containing chromosome-specic markers (Table 1).
BAC DNA labelling, hybridizations and signal detection were carried out according to standard protocols
(Raudsepp and Chowdhary 2008). The results were
examined with Zeiss Axioplan2 uorescence microscope, and at least 10 images were captured and
analysed for each experiment using ISIS V5.2 (MetaSystems GmbH) software.

Table 1. Markers used for FISH analysis; EquCab2 horse reference genome (UCSC Genome Browser http://genome.ucsc.edu/)
Gene/marker symbol
NPY
ASB22
UMNe104
PEX7
URI1
PRKAG2

(a)

Genomic position, EquCab2

Cytogenetic location

CHORI-241 BAC

4:55,708,297-55,715,462
4:59,506,355-59,506,518
4:64,143,130-64,143,285
10:82,269,103-82,357,588
10:1,696,921-1,723,107
4:103,079,760-103,357,356

4q21.1-q21.3
4q21
4q21.1-q21.3
10q22-q23
10p15
4q26-q27

42L1
158I3
141L20
83E10
40B19
109O22

Reference
Raudsepp et al.
This study
Raudsepp et al.
Lear et al. 2001
Raudsepp et al.
Raudsepp et al.

2008
2008
2008
2008

(b)

(c)

Fig. 1. Cytogenetic characterization of the translocation. (a) A GTG-banded metaphase spread and (b) corresponding karyogram; (c) GTGbanded normal and aberrant chr4 and chr10 from four dierent cells; dotted line indicates the translocation breakpoint

2015 Blackwell Verlag GmbH

Autosomal Translocation and Embryonic Loss in a Mare

(a)

173

(b)

(c)

(d)

(e)

Fig. 2. Translocation analysis by FISH. (ad) inverted DAPI-stained normal and aberrant chr4 and chr10 after FISH with selected markers (see
Table 1 for details); (e) schematic drawings of normal and aberrant chr4 and chr10; cytogenetic locations of six selected markers are shown to the
right of ideograms; deleted segment in chr4 is demarcated with red dotted line; translocation of 4q21-qter is indicated with a red arrow and shown
as red/white ideogram

Results
Initial karyotyping of Giemsa-stained metaphases
revealed 64 chromosomes, including two normal X
chromosomes. The latter was conrmed by CBG banding. However, in each cell we observed the presence of
two derivative submetacentric chromosomes: one very
small and another abnormally large. Further analysis by
GTG banding determined that the small chromosome
was chr4 that was missing a large segment of the long (q)
arm, and the large chromosome was composed of the
missing part of chr4q and the entire chr10 (Fig. 1a,b).
Analysis of 20 GTG-banded cells conrmed the presence
of the translocation in all cells and indicated that the
translocation break and fusion points were approximately at 4q21 and 10p15, respectively (Fig. 1c).

The involvement of chr4 and chr10 in the translocation

was conrmed by FISH using BAC clones containing
PRKAG2 from chr4q26-q27 and PEX7 from 10q22-q23
(Fig. 2a; Table 1). To determine whether the exchange
was reciprocal and demarcate translocation breakpoint
(s), additional BACs containing NPY, ASB22 and
UMNe104 from chr4q21.1-q21.3 and URI1 from
chr10p15 (Table 1) were used for FISH. In chr4, the
breakpoint was localized between NPY and UMNe104
(Fig. 2b) and further narrowed down to an approximately 4.6-Mb region (chr4:59,506,518-64,143,130)
between ASB22 and UMNe104 (Fig. 2c; Table 1). The
translocation appeared to be non-reciprocal because
URI1 from chr10p15 was present in both the normal
chr10 and the one fused with chr4q (Fig. 2d).

Table 2. Summary data about balanced autosomal translocations in horses with REEL
Karyotype
64,XX,t(1q;3q)
64,XY,t(1;30)
64,XX,t(1;16)
64,XX,t(1;21)
64,XX,t(16;22)
64,XX,t(4;13)
64,XY,t(5;16)+mar
64,XX,t(2;13)
64,XX,t(4;10)

Breakpoints
q14;q13
pter;qcen
q16;q21.3
q14;qter
q13;q18
pqcen;pqcen
qcen;p17
p13;pter
q21;p15

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Type

Breed

Reference

Reciprocal
Nonreciprocal
Reciprocal
Nonreciprocal
Reciprocal
Reciprocal
Nonreciprocal
Nonreciprocal
Nonreciprocal

Thoroughbred
Thoroughbred
Thoroughbred
Thoroughbred
Thoroughbred
Thoroughbred
Thoroughbred
Thoroughbred
Arabian

Power 1991
Long 1996
Lear and Layton 2002
Lear et al. 2008
Lear et al. 2008
Lear et al. 2008
Durkin et al. 2011
Lear et al. 2014
This study

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S Ghosh, PJ Das, F Avila, BK Thwaits, BP Chowdhary and T Raudsepp

We concluded that the Arabian mare carried a nonmosaic and non-reciprocal balanced autosomal translocation t(4;10)(q21;p15), which was the most likely cause
of REEL.

Discussion
We characterized a novel autosomal translocation
t(4;10)(q21;p15) in the horse and reported a new case
in which subfertility due to REEL was associated with
chromosomal abnormality. While eight similar cases
have been previously described in Thoroughbred horses
(Table 2), this is the rst found in Arabians. As we had
no access to fertility records or samples for the dam and
sire of the case, we cannot speculate on whether this was
a de novo or inherited condition.
Examination of all REEL-associated translocations in
horses (Table 2) shows that none is recurrent. Even
though chrs1, 4, 13 and 16 are involved more than once,
chromosome combinations and translocation breakpoints are dierent in each case (Table 2). On the other
hand, only 11 of the 31 equine autosomes have been
engaged in these translocations which might be due to
very few available reports or because these equine
chromosomes are more prone for rearrangements.
Evidence for the latter is sparse (Lear et al. 2008), and

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the breakpoint at chr4q21 in this study showed no

overlap with any known evolutionary breakpoint
(Murphy et al. 2005) or region of genome instability
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carriers.
Conflict of interest
The authors have declared that no competing interests exist.

Author contributions
Clinical data and samples were provided by BKT; the study was
designed by TR and BPC; experiments were conducted, analysed and
interpreted by SG, PJD, FA and TR, and the manuscript was drafted
by TR, SG, PJD, FA and BPC.

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Submitted: 11 Sep 2015; Accepted: 6 Oct
2015
Authors address (for correspondence): T
Raudsepp, Department of Veterinary Integrative Biosciences, College of Veterinary
Medicine & Biomedical Sciences, Texas
A&M University, College Station, TX
77843-4458, USA. E-mail: traudsepp@cvm.
tamu.edu

2015 Blackwell Verlag GmbH