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Tierärztliche Hochschule Hannover
Population and molecular genetic analyses
of persistent right aortic arch and primary cataracts
in the German Pinscher
INAUGURAL-DISSERTATION
zur Erlangung des Grades einer Doktorin der Veterinärmedizin
- Doctor medicinae veterinariae –
(Dr. med. vet.)
vorgelegt von
Julia Menzel
Stuttgart
Hannover 2010
Wissenschaftliche Betreuung: Prof. Dr. Ottmar Distl
Institut für Tierzucht und Vererbungsforschung
1. Gutachter: Prof. Dr. Ottmar Distl
2. Gutachter: Prof. Dr. Sabine Kästner
Tag der mündlichen Prüfung: 20.05.2010
Teile dieser Arbeit sind bei folgenden Zeitschriften zur Veröffentlichung
angenommen:
1. The Veterinary Journal
2. Veterinary Ophthalmology
Table of contents
1 Introduction 01
2 Unusual vascular ring anomaly associated with a
persistent right aortic arch and an aberrant left subclavian
artery in German pinschers 05
2.1 Abstract 06
2.2 Introduction 06
2.3 Material and Methods 08
2.4 Results 10
2.5 Discussion 13
2.6 Conclusions 15
2.7 Conflict of interest statement 16
2.7 Acknowledgements 16
2.8 References 17
2.9 Appendix 20
3 Evaluation of the canine TBX1 gene as candidate for
a rare form of persistent right aortic arch in the German
Pinscher 25
3.1 Abstract 26
3.2 Introduction 26
3.3 Material and Methods 28
3.4 Results and Discussion 30
3.5 Acknowledgements 32
3.6 References 33
3.7 Appendix 35
4 Prevalence and formation of primary cataracts in the
German Pinscher population in Germany 51
4.1 Abstract 52
4.2 Introduction 53
4.3 Material and Methods 54
4.4 Results 56
4.5 Discussion 58
4.6 Acknowledgements 60
4.7 References 61
4.8 Appendix 64
5 Scanning 20 candidate genes for association with primary
cataracts in the German Pinscher population in Germany 67
5.1 Abstract 68
5.2 Introduction 68
5.3 Material and Methods 69
5.4 Results and Discussion 73
5.5 Acknowledgements 74
5.6 References 75
5.7 Appendix 78
6 General Discussion 93
7 Summary 101
8 Erweiterte Zusammenfassung 105
9 Appendix 123
10 List of publications 135
11 Acknowledgements 137
List of Abbreviations
A Adenine
ARVCF armadillo repeat gene deletes in velocardiofacial syndrome
BFSP2 beaded filament structural protein 2, phakinin
BLAST basic local alignment search tool
BLASTN basic local alignment search tool nucleotide
bp base pairs
C cytosine
CAT primary non-congenital cataract
CDC45L cell division cycle 45 homolog
cDNA complementary deoxyribonucleid acid
CFA canis familiaris autosome
CLTCL1 clathrin, heavy chain-like 1
COMT catechol-O-methyltransferase
CRYAA crystallin, alpha A
CRYAB crystallin, alpha B
CRYBA1 crystallin, beta A1
CRYBB2 crystallin, beta B2
CRYGA crystallin, gamma A
DGCR DiGeorge crirical region
DNA deoxyribonucleid acid
DOK Dortmunder Kreis, German panel of the European Eye Scheme
for diagnosis of inherited eye diseases in animals
ECV European College of Veterinary Ophtalmologists
EDTA ethylenediamine tetraacetic acid
EYA1 eyes absent homolog 1
DGS14 DiGeorge syndrome critical region protein 14
DMSO dimethyl sulfoxide
DNA deoxyribonucleid acid
dNTPs deoxy nucleoside 5’triphosphates (N is A,C,G or T)
F forward
FOXE3 forkhead box E3
FTL ferritin, light polypeptide
G guanine
GCNT2 glucosaminyl (N-acetyl) transferase 2, l-branching enzyme
GJA3 gap junction protein, alpha 3, 46kDa (connexion 46)
GJA8 gap junction protein, alpha 8, 50kDa (connexion 50)
GSC2 goosecoid homebox 2
HET heterozygosity
HIRA histone cell cycle regulation defective homolog A
HSF4 heat shock transcription factor 4
Indel insertion/deletion
IRD infrared dye
kb kilobase
LIM2 lens intrinsic membrane protein 2, 19 kDa
LOD logarithm of the odds
M molar
MAF v-maf musculoaponeurotic fibrosarcoma oncogene homolog
Mb mega base
MERLIN multipoint engine for rapid likelihood inference
MIP major intrinsic protein of lens fiber, aquaporin
MRPL40 mitochondrial ribosomal protein L40
MS microsatellite
NCBI National Center for Biotechnology Information
no. number
NPL nonparametric linkage
P error probability
PAX6 paired box gene 6
PCR polymerase chain reaction
PIC polymorphism information content
PIED presumed inherited eye diseases
PHTVL persistent hyperplastic tunica vasculosa lentis
PITX3 paired-like homeodomain transcription factor 3
POS position
PRAA persistent right aortic arch
PRAA-SA persistent right aortic arch with an aberrant left subclavian
artery
PRAA-SA-LA persistent right aortic arch and left retrooesophageal subclavian
artery in combination with a ligamentum arteriosum originating
at the aberrant left subclavian artery
PSK Pinscher-Schnauzer-Klub 1895 e.V.
R reverse
RNA ribonucleid acid
SAA subclavian artery anomalies
SAS statistical analysis system
SIX5 sine oculis homeobox homolog 5
SLC25A1 solute carrier family 25 member 1
SNP single nucleotide polymorphism
SORD sorbitol dehydrogenase
T thymine
Ta annealing temperature
TBX1 t-box gene 1
TE tris-ethylenediamine tetraacetic acid
TRNT1 tRNA nucleotidyl transferase, CCA-adding, 1
TXNRD2 thioredoxin reductase 2
UFD1 ubiquitin fusion degradation protein
UV ultraviolet
VRA vascular ring anomaly
CHAPTER 1
Introduction
Introduction
2
1 Introduction
The German Pinscher is an old breed and is included in the origins of the Doberman
Pinscher, the Miniature Pinscher, the Affenpinscher, the Miniature Schnauzer, the
Standard Schnauzer and the Giant Schnauzer. The Wired Haired and Smooth Haired
Pinschers, as the Standard Schnauzer and German Pinscher were originally called,
were shown in dog books as early as 1884. Following both World Wars, the breed
was nearly lost. There were no new litters registered in West Germany from 1949 to
1958. At this time, Werner Jung searched the farms in Germany for typical Pinschers
and used these dogs along with four oversized Miniature Pinschers and a black and
red bitch from East Germany. Most German Pinschers today are descendants of
these dogs.
The modern German Pinscher has therefore a relatively small gene pool. Attention to
potential health concerns is very important for the breed in the future. With increasing
knowledge on prevalence and pathogenesis of eye diseases and vascular ring
anomalies, breeding guidelines need to be developed for reducing the prevalences of
presumed inherited diseases. Therefore, it is of particular importance to clarify the
population and molecular genetic background of these diseases.
Vascular ring anomalies (VRAs) are developmental anomalies of the embryonic
aortic arches. Persistent right aortic arch (PRAA) is the most common VRA in dogs.
Because vascular rings cause oesophageal compression, regurgitation soon after
eating solid food is the principal clinical sign of PRAA in young dogs. In PRAA-
affected dogs, the aorta is formed by the right fourth aortic arch instead of the left
fourth aortic arch. In the German Pinscher, a rare combination of anomalies occurs.
This type of PRAA (PRAA-SA-LA) is characterized by a left retrooesophageal
subclavian artery in combination with a ligamentum arteriosum originating at the
aberrant left subclavian artery, and has only been reported in two isolated cases of
other dog breeds before. Surgical treatment is the only effective treatment for this
disease, and therefore prevention is very important for animal welfare.
Primary cataracts are characterized as a focal or diffuse opacity of the eye lens. It is
a very common eye disease in the dog, and reported prevalences range between 1.8
Introduction
3
and 88.0%. The age of onset of inherited cataracts may be congenital, juvenile or
senile. Usually inheritance is presumed, based on the typical appearance and age in
a breed known to be predisposed to cataracts. In the majority a recessive mode of
inheritance is existent, but also dominant pattern are described. Given the limited
success of medical treatment and the invasiveness of surgical treatment of cataracts,
prophylactive measures should be considered more closely. At this time, many
kennel clubs have developed selection programs to reduce the prevalences of
primary cataracts in their breed.
In the future, the most successful method to reduce primary cataracts as well as
PRAA would be to identify the genetic background and the causal mutations of both
diseases in the affected breeds. Genetic tests can be used to select breeding
animals that do not carry and transmit defect alleles. Especially in a small population
as the German Pinscher is, a DNA test, showing if the dog is homozygous for a
primary cataract-causing or PRAA-causing mutation or a heterozygous carrier or free
from primary cataract-causing or PRAA-causing mutations would be very helpful.
Combined with an adequate breeding program, the prevalence of primary cataracts
and PRAA could be more effectively decreased, and in addition, the risk of a
selection-caused bottle-neck-phenomenon could be minimized.
The purpose of this study was to analyse the population and molecular genetic
background of primary cataracts and persistent right aortic arch in German Pinschers
and to describe a specific form of persistent right aortic arch which has only been
reported in two isolated cases of other dog breeds before.
The contents of the present thesis are presented in single papers as allowed by § 8
(3) of the Rules of Graduation (Promotionsordnung) of the University of Veterinary
Medicine Hannover, Germany. The report of the specific form of persistent right
aortic arch in German Pinschers is presented in chapter 2, while chapter 3 comprises
the results of evaluation of TBX1 as a candidate gene for this rare form of persistent
right aortic arch in German Pinschers. The study of prevalence and formation of
primary cataracts in the German Pinscher population in Germany is presented in
chapter 4. The results of molecular genetic analyses of primary cataracts in German
Introduction
4
Pinschers are presented in chapter 5. Finally, the results of the present thesis are
generally discussed and summarised in chapter 6 to 8.
CHAPTER 2
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery
in German Pinschers
Julia Menzel, Ottmar Distl
Institute for Animal Breeding and Genetics, University of Veterinary Medicine
Hannover, Foundation, Bünteweg 17p, 30559 Hannover, Germany
Accepted for publication in: The Veterinary Journal
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
6
2 Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
2.1 Abstract
The objective of this study was to describe a specific form of persistent right aortic
arch (PRAA) in three German Pinschers and to analyse the mode of inheritance in
this dog breed. This type of PRAA is characterized by a left retrooesophageal
subclavian artery in combination with a ligamentum arteriosum originating at the
aberrant left subclavian artery (PRAA-SA-LA). This rare combination of anomalies
has only been reported in two isolated cases of other dog breeds before. In the
German Pinscher, the occurrence of any form of PRAA was not previously known. In
this study, 18 cases of this congenital anomaly were ascertained and their high
degree of relatedness and inbreeding could be shown through pedigree analysis.
Three of the affected dogs underwent further clinical investigations and for two of
them, post-mortem findings and, for one dog, findings at the surgery verified the
diagnosis of PRAA-SA-LA. In this third dog, the PRAA-SA-LA was successfully
corrected by surgery and after this intervention normal development was observed. A
monogenic autosomal recessive mode of inheritance was not likely. Further research
is required for unravelling a possible involvement of genes located within the syntenic
canine DiGeorge region.
2.2 Introduction
Vascular ring anomalies (VRAs) are developmental anomalies of the embryonic
aortic arches. The result of these anomalies is a complete or partial circle around the
trachea and the oesophagus formed by blood vessels and associated structures,
which may lead to a compression of the encircled structures. Persistent right aortic
arch (PRAA) is recognized as the most common VRA (Helphrey, 1979; Ellison, 1980;
Muldoon et al., 1997), representing 95 % of all VRAs diagnosed in dogs (Buchanan,
2004). In PRAA affected dogs, the aorta is formed by the right fourth aortic arch
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
7
instead of the left fourth aortic arch (Kim et al., 2006). PRAA is a common cause of
regurgitation and usually results in a dilated oesophagus in young dogs (Muldoon et
al., 1997, Kim et al., 2006). In this anomaly, the ligamentum arteriosum typically joins
the pulmonary artery to the abnormally positioned aorta and creates the VRA around
the oesophagus and the trachea. PRAA with an aberrant left subclavian artery
(PRAA-SA) is observed in 33% of all dogs with PRAA (Buchanan, 2004). In cases of
a PRAA-SA, the oesophagus is compressed by the vascular ring comprised of the
persistent right aortic arch with left subclavian artery and by a partial ring formed by
the aberrant left subclavian artery (Ellison, 1980). Only in two reported cases of
PRAA-SA, the ligamentum arteriosum extended from the main pulmonary artery to
the aberrant left subclavian artery instead of the aortic arch (PRAA-SA-LA) (House et
al., 2005). This rare course of the ligamentum arteriosum enforces the compression
of the oesophagus by the aberrant left subclavian artery.
Initially, no clinical signs are visible in the newborn puppies affected by PRAA,
PRAA-SA or PRAA-SA-LA. The first clinical symptoms appear with the ingestion of
solid food and consist of postprandial regurgitation of food in both types of vascular
ring anomalies (Buchanan, 2004; VanGundy, 1989). Because of the constriction of
the oesophagus at the level of the heart base, transport of ingesta is not possible or
markedly decreased, leading to regurgitation. In addition, part of the ingested food
remains cranial to the constriction in the oesophagus, resulting in enlargement of the
oesophagus in the precardiac area. Affected puppies retard in growth and lose
weight despite great appetite. Contrast radiographies of the thorax show
oesophageal dilatation cranial to the heart with constriction at the level of the heart
base (Buchanan, 1968; VanGundy, 1989). The extent of the oesophageal
constriction varies with some puppies not able to ingest paste-like food and others
puppies regurgitating only after solid food intake.
Surgical treatment is required to eliminate the oesophageal constriction (Ellison,
1980). The postoperative treatment includes feeding slurry of canned food in a
vertical position. The earlier the diagnosis and the surgical intervention, the better are
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
8
the chances for complete recovery and the absence of a precardiac dilatation of the
oesophagus (Ellison, 1980; VanGundy, 1989; House et al., 2005; Fingeroth and
Fossum, 1987). Reports of long-term outcome following surgical correction are
variable: a recent study found that 92% of the dogs had a complete resolution of
clinical signs even though mild cranial oesophageal dilation persisted (Muldoon et al.,
1997). Epidemiologic studies as well as breeding studies have shown that German
Shepherds, Irish Setters and Greyhounds are genetically predisposed to the
development of PRAA (Patterson, 1968; Gunby et al., 2004), the mode of inheritance
being complex and polygenic in its basis (Patterson, 1989).
There are no reports of any forms of PRAA in German Pinschers. However, in the
last 6 months PRAA-SA-LA has been diagnosed in three German Pinscher puppies
from Germany and The Netherlands. Fifteen other cases of PRAA have been
reported by breeders or veterinarians and therefore have to be regarded partly as
presumptive diagnoses. This number of cases shows a high prevalence in German
Pinschers and raises the suspicion of a genetic cause. The objectives of this study
were to describe the congenital conotruncal anomalies and to examine if PRAA could
be an inherited defect in German Pinschers.
2.3 Materials and methods
The Pinscher-Schnauzer-Klub (PSK) in Germany contacted the Institute for Animal
Breeding and Genetics of the University of Veterinary Medicine, Hannover because
of several recent cases of PRAA in German Pinschers. A total of 18 cases from 16
different litters were reported in the last ten years. Three of them underwent further
investigations at the Hannover Institute for Animal Breeding and Genetics.
In the three German Pinschers, the cause of oesophageal constriction was a
persistent right aortic arch, an aberrant left subclavian artery and a left ligamentum
arteriosum with its origin on the left subclavian artery (PRAA-SA-LA). The 15
remaining cases were reported by veterinarians or breeders and dog owners from
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
9
Germany and the Netherlands. One of these 15 puppies was examined in a private
institute for pathology in the Netherlands with the diagnosis of PRAA-SA-LA. Another
dog underwent corrective surgery at the Faculty of Veterinary Medicine at the
Ludwig-Maximilians-University in Munich ten years ago with a diagnosis of PRAA
without further specification and lives now without any complications. In the other 13
dogs, diagnoses were made on the basis of clinical signs, radiographies and contrast
oesophagrams, and therefore have to be regarded as presumptive diagnoses. One
female dog which is now seven years old lives since the age of eight months with the
presumptive diagnosis of PRAA (not specified). As the owner reported, this PRAA-
affected dog was the smallest puppy of the litter and always a bit too thin. The clinical
signs started soon after weaning with intermitted postprandial regurgitation though
showing a great appetite. The veterinarian made the presumptive diagnosis of PRAA
using radiographies, clinical symptoms and contrast oesophagram. The owner
started to feed this dog about six to eight times daily with small amounts of slurry of
pureed soft dog food mixed with water in an upright position after this time and kept
this feeding regime up to now. The dog is doing more or less well with this treatment,
regurgitation occurs at a maximum of two times/month, mostly after having obtained
pieces of normal dog food or something similar. At present, the dog has a normal
weight for his size. All in all, 2/18 dogs ascertained underwent a corrective surgery,
15/18 dogs were euthanized as puppies mainly because of poor body condition, and
1/15 dog is living without surgical treatment.
The pedigrees of the affected dogs were provided by the PSK and were used for
segregation analysis to test whether the data were compatible with the respective
simple Mendelian model, a recessive mode of inheritance. The Singles method is a
very straightforward method of simple segregation analysis (Davie, 1979). The test
involves a statistical comparison of the estimated segregation frequency P
(probability that an offspring is affected by the respective disease) with the
hypothesized value (P0) arising from the particular model of inheritance being tested.
If a recessive mode of inheritance is assumed, both parents in each of a set of full sib
families are unaffected, the null hypothesis is that the true value of P0 = 0.25. The
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
10
most straightforward use of the Singles method can be made when the investigator is
certain that all families with affected offspring are included in the data. Then the
segregation frequency can be estimated as P = (A - A1) / (T - A1) and its estimated
variance is given by Est. Var. (P) = (T - A) / (T - A1)3 [A - A1 + 2A2 (T - A) / (T - A1)]
where A is the total number of affected offspring in the available data, T is the total
number of all examined offspring in the available data, A1 is the total number of
families with just one affected offspring, and A2 is the total number of families with
two affected offspring. Then the null hypothesis is tested using: Z2 = (P - P0)2 / Est.
Var. (P). If the calculated value Z2 is not significant at α = 0.05, the data are
consistent with a simple recessive mode of inheritance.
The mean coefficient of relationship was calculated using OPTI-MATE (Wrede and
Schmidt, 2003) for the group of German pinschers with PRAA affected offspring (n =
27) and a contemporary group of dogs born in 2008 (n = 555). The mean coefficient
of inbreeding was compared among PRAA affected dogs and the same
contemporary group of dogs. Pedigree information over eight generations was
considered, the completeness of pedigrees was larger then 95% in all eight
generations. The mean number of puppies per litter was calculated for the group of
litters with PRAA affected offspring (n = 16) and a contemporary group of all litters in
2005 – 2009 (n = 415). P values were calculated using t tests.
2.4 Results
Case reports
The first actual case was a three week-old female German Pinscher puppy (puppy 1)
of a German breeder which was euthanized because of constant regurgitation and
poor body condition. Contrast oesophagram with barium sulphate made by the
referral veterinarian revealed a precardiac mega oesophagus and oesophageal
constriction at the level of the heart base. After euthanasia, puppy 1 was presented
to the Hannover Institute for Pathology to investigate the cause of oesophageal
constriction. A severe dilatation of the oesophagus cranial to the heart base and
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
11
constriction at the level of the heart base was found. The diameter of the oesophagus
cranial to the heart was four times larger than that at the level of the heart base. The
oesophagus was present between a persistent right aortic arch, a left subclavian
artery, the heart base and the ligamentum arteriosum which extended from the main
pulmonary artery to the left subclavian artery, attaching approximately one cm cranial
to the point of origin of the aberrant left subclavian artery. The main cause of the
constriction seemed to be the aberrant left subclavian artery in combination with the
ligamentum arteriosum, the aortic arch made only a minor contribution to the
constriction.
In cases 2 and 3, a Dutch breeder of German Pinschers contacted the Hannover
Institute for Animal Breeding and Genetics because of two suspected PRAA affected
male puppies (puppy 2 and 3) in one of their litters. The referral veterinarian had
made radiographies and contrast oesophagrams of both puppies at an age of four
weeks and suspected PRAA because of the radiographic findings and clinical signs
(postprandial regurgitation and growth retardation). The two puppies were presented
to the Hannover Small Animal Clinic where a complete clinical examination,
radiographies, contrast oesophagrams, and ultrasonography of the heart were
performed. The contrast oesophagram of puppy 2 showed the oesophagus highly
dilated cranial to the heart base (Fig. 1), the contrast oesophagram of puppy 3
showed only a mild dilatation cranial to the heart base. In the ultrasonographic
examination of puppy 2, a ventricle septum defect was also diagnosed. The
ultrasonographic examination of puppy 3 revealed no abnormalities.
Because of the complicated medical findings in puppy 2 and the poor prognosis for
the ventricle septum defect, puppy 2 was euthanized and sent to post-mortem
examination. Puppy 3 was submitted to a surgical treatment on the next day. The
thoracic cavity was entered through a left lateral thoracotomy incision at the level of
the fourth left intercostal space. The oesophagus was found compressed by a
complete ring formed with a aberrant persistent left ligamentum arteriosum joining
the left subclavian artery and the pulmonary artery and their left subclavian artery.
After ligation and transsection of the ligamentum arteriosum, the oesophagus was no
more compressed by the aberrant left subclavian artery; therefore this vessel was not
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
12
manipulated. One day after surgery, the dog looked alert and eager to eat. Feeding
in an upright position was resumed from the second postoperative day with small
amount of a slurry of soft dog food 7-8 times daily, after then the dog was fed with
small amounts of normal soft dog food mixed with a little water 4-5 times daily in a
normal position. No episodes of regurgitation were noticed neither during the upright
position feeding interval nor the feeding interval at a normal position. Six weeks after
the surgery, the puppy lives at his new owner family, and no problems like
regurgitation have occurred since the surgical treatment. He gained weight quickly
and is now nearly as big as his littermates.
In the autopsy of puppy 2 (Fig. 2 + 3), the diagnostic findings were very similar to the
findings in puppy 1. The oesophagus was found highly dilated in the precardiac
region; the diameter in this region was five times larger than at the level of the heart
base. The postcardiac region showed no abnormalities. The oesophagus was found
in the same position as in puppy 1, between a persistent right aortic arch, a left
aberrant subclavian artery, the heart base and the ligamentum arteriosum which
extended again from the main pulmonary artery to the left subclavian artery,
attaching 1.5 cm cranial to the point of origin of the aberrant left subclavian artery.
The aberrant left subclavian artery in combination with the ligamentum arteriosum
seemed to be the main cause of constriction. The persistent right aortic arch was
also involved in the constriction, but only in a minor degree.
Pedigree analysis
Investigation of the pedigrees showed relationships among all 18 affected dogs (Fig.
4). The mean coefficient of inbreeding of PRAA affected German Pinschers was 6.76
% which is significantly higher than the mean coefficient of inbreeding of the
contemporary group (3.56 %, Table 1). The mean coefficient of relationship among
parents with at least one PRAA affected progeny was with a value of 11.04 % higher
than the mean coefficient of relationship of the contemporary group (7.44 %). The
mean number of puppies per litter calculated for the group of litters with PRAA
affected offspring was 6.20 ± 2.33 which was not significantly different from the mean
number of puppies per litter calculated for the contemporary group (6.47 ± 2.59). All
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
13
affected dogs had unaffected parents and the proportion of affected males (n = 8)
and females (n = 7) was almost equal (sex unknown: n = 3). Therefore, an X-linked
mode of inheritance was excluded as well as a monogenic autosomal dominant
mode and a mitochondrial mode. The result of the simple segregation analysis using
the Singles method revealed a Z2 of 29.51 which was significant at α = 0.05.
Therefore the observed distribution of PRAA was not consistent with a simple
recessive mode of inheritance.
2.5 Discussion
The left retrooesophageal subclavian artery in combination with the left ligamentum
arteriosum with its origin on the left subclavian artery was the main cause of
oesophageal constriction in the examined three German Pinscher puppies. Even if
the other cases reported could not be verified in post-mortem examinations, the close
relationships among the cases suggest that the same VRA may have caused the
dilatation of the oesophagus. The extremely rare combination of anomalies that
occurred in the three German Pinscher puppies has been described in dogs in only
two cases before, in one German Shepherd dog and one Great Dane (House et al.,
2005). This combination of anomalies is similar to an inherited anomaly reported in
humans in whom a diverticulum is present in addition at the origin of the left
subclavian artery (Kommerell’s diverticulum) (Cina et al., 2000).
The only definitive treatment for VRAs is surgery. Medical therapy alone without
surgery, consisting of feeding in an upright position and slurry diets, is considered as
not being effective and in most cases leads to worsening of oesophageal dilation and
aspiration pneumonia. Nevertheless, the seven year-old female German Pinscher
reported here lives since the age of eight months with the presumptive diagnosis of
PRAA. The dog has a normal weight for his size and aspiration pneumonia never
appeared until today.
The six most commonly reported heart defects in dogs are patent ductus arteriosus,
pulmonic stenosis, subaortic stenosis, ventricular septal defect, tetralogy of Fallot,
and PRAA. All of them have been proven to be heritable through genetic studies
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
14
(Patterson 1989). In the case of PRAA, breeding studies (German Shepherds) as
well as epidemiologic studies (German Shepherds, Irish Setter) have demonstrated
that German Shepherds and Irish Setters are genetically predisposed to its
development; and that the mode of inheritance of PRAA is likely complex and
polygenetic (Patterson, 1968; Patterson, 1989). Investigation of the heritability of
congenital heart diseases started with epidemiologic studies at first; the results
indicated that the number of PRAA affected dogs in the German Shepherd and Irish
Setter breeds is significantly higher than in other breeds. In breeding studies
involving matings between two German Shepherd dogs with PRAA, there was a
higher frequency of this anomaly in the offspring, and the type of PRAA in those
puppies was identical or closely related to that of the parents (Patterson, 1968).
The close relationships and high inbreeding coefficients suggest that PRAA in
German Pinschers is an inherited defect. The higher the coefficient of inbreeding, the
more likely an inherited recessive defect may be evident in the offspring. The
coefficient of inbreeding of the group of German Pinschers with PRAA affected
offspring is significantly higher than in the contemporary group. We could not
determine the exact mode of inheritance for PRAA in German Pinschers. Using the
Singles method for segregation analysis, a monogenic autosomal recessive
inheritance could be rejected. The proportion of affected puppies per litter was clearly
below 0.25 and only in two litters this proportion was close to 0.25 (2/7 = 0.286 and
2/9 = 0.22). Even differences in litter sizes among litters with PRAA affected and
without PRAA affected puppies were small and did not seem to influence the
segregation ratio of affected individuals. Therefore, this mode of inheritance did not
appear plausible. Thus, an oligogenic or polygenic mode of inheritance or the
contribution of a larger number of mutations of a genomic region appeared to be
more likely.
Comparative genetic research revealed that conotruncal heart defects in humans
have been associated with deletion of the chromosome 22q11.2 region which is also
known as the DiGeorge critical region (DGCR) (Rauch et al., 2004; Lee et al., 2006).
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
15
Monosomy 22q11.2 was found in 46% of patients with a PRAA but in only 30% of
patients with left aortic arch (Rauch et al., 2004). The microdeletion was also found in
81% of patients with, but only in 17% of patients without subclavian artery anomalies
(SAAs) (Rauch et al., 2004). Thus, the incidence of monosomy 22q11.2 was
associated rather with the presence of SAAs than with the laterality of the aortic arch
(Rauch et al., 2004). The 3-Mb interval encompassing the DGCR contains 30 genes
that are deleted in patients with 22q11.2 deletion syndrome (del22q11.2) which
includes diagnoses of DiGeorge syndrome, velo-cardio-facial syndrome and
conotruncal anomaly face syndrome. Cardiovascular anomalies found in these
patients include tetralogy of Fallot, aortic-arch anomalies, persistent truncus
arteriosus and ventricular septal defects. The genes located within the DGCR on
22q11.2 are almost completely conserved on mouse chromosome 16. Mice with a
heterozygous deletion of a 1.5-Mb homologous DiGeorge region show defects similar
to those seen in del22q11.2 patients. Among the T-box genes important for
cardiomorphogenesis, the TBX1 gene is located in the DGCR. TBX1-/- mice exhibited
many of the cardiovasular malformations seen in del22q11.2 syndrome patients
(Lindsay et al., 2001). These observations made TBX1 a candidate gene for the
cardiovascular manifestations of del22q11.2 syndrome. However, only three TBX1
mutations have been identified that accounted for < 1% of conotruncal malformations
in these populations (Gong et al., 2001; Yagi et al., 2003; Stoller et al., 2005).
A study in which comparative mapping of the DGCR region in the dog (Keeshond)
has been performed revealed that this region mapped to the telomeric end of
chromosome 26 and appeared to be conserved in the dog (Werner et al., 1999).
However, in the same study linkage was not evident between conotruncal heart
defects in Keeshonds and the canine gene loci mapped within this region.
2.6 Conclusions
Monosomy 22q11.2 is strongly associated with the presence of SAAs which were
found in all three puppies examined. Linkage analyses of canine gene loci mapped in
the DiGeorge region with PRAA in German pinschers could help clarify whether
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
16
mutations in this region are responsible for this anomaly. The reported German
Pinscher families may be useful to unravel genes involved in PRAA-LA-SA. 2.7 Conflict of interest statement
None of the authors of this paper has a financial or personal relationship with other
people or organisations that could inappropriately influence or bias the content of the
paper. 2.8 Acknowledgements
We thank the Pinscher-Schnauzer-Klub e. V. (PSK) in Germany for support in
collecting pedigrees and cases of PRAA and all German Pinscher breeders for
providing data and diagnoses from their dogs.
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
17
2.9 References
Buchanan, J.W., 2004. Tracheal signs and associated vascular anomalies in dogs
with persistent right aortic arch. Journal of Veterinary Internal Medicine 18, 510-
514.
Buchanan, J.W., 1968. Patent ductus arteriosus and persistent right aortic arch
surgery in dogs. Journal of Small Animal Practice 9, 409-428.
Cina, C.S., Arena, G.O., Bruin, G., Clase, C.M., 2000. Kommerell’s diverticulum and
aneurismal right sided aortic arch: a case report and review of literature. Journal
of Vascular Surgery 32, 1208-1214.
Davie, A.M., 1979. The singles method for segregation analysis under incomplete
ascertainment. Annals of Human Genetics 42, 507-512.
Ellison, G.W., 1980. Vascular ring anomalies in the dog and cat. Compendium on
Continuing Education for the Practicing Veterinarian 2, 693-705.
Fingeroth, J.M., Fossum, T.W., 1987. Late-onset regurgitation associated with
persistent right aortic arch in two dogs. Journal of the American Veterinary
Medical Association 191, 981-983.
Gong, W., Gottlieb, S., Collins, J., Blescia, A., Dietz, H., 2001. Mutation analysis of
TBX1 in non-deleted patients with features of DiGeorge
Syndrom/Velocardiofascial Syndrom or isolated cardiovascular defects. Journal
of Medical Genetics 38, E45.
Gunby, J.M., Hardie, R.J., Bjorling, D.E., 2004. Investigation of the potential
heritability of persistent right aortic arch in Greyhounds. Journal of the American
Veterinary Medical Association 224, 1120-1121.
Helphrey, M.L., 1979. Vascular ring anomalies in the dog. Veterinary Clinics of North
America 9, 207-218.
House, A.K., Summerfield, N.J., German, A.J., Noble, P.J.M., Ibbarola, P.,
Brockmann D.J., 2005. Unusual vascular ring anomaly associated with a
persistent right aortic arch in two dogs, Journal of Small Animal Practice 6, 585-
590.
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
18
Kim, N.S., Alam, M.R., Choi, I.H., 2006. Persistent right aortic arch and aberrant left
subclavian artery in a dog: a case report. Veterinarni Medicina 51, 156-160.
Lee, M.-L., Chen, H.-N., Chen, M., Tsao, L.-Y., Wang, B.-T., Lee, M.-H., Chiu, I.-S.,
2006. Persistent fifth aortic arch associated with 22q11.2 deletion syndrome.
Journal of the Formosan Medical Association 105, 284-289.
Lindsay, E.A., Vitelli, F., Su, H., Morishima, M., Huynh, T., 2001. Tbx1
haploinsufficiency in the DiGeorge syndrome region causes aortic arch defects in
mice. Nature 410, 97-101.
Muldoon, M, Birchard, S.J., Ellison, G.W., 1997. Long-term results of surgical
correction of persistent right aortic arch in dogs: 25 cases (1980-1995). Journal of
the American Veterinary Medical Association 210, 1761-1763.
Patterson, D.F., 1968. Epidemiologic and Genetic Studies of Congenital Heart
Disease in the dog. Circulation Research 23, 171-2002.
Patterson, D.F., 1989. Hereditary congenital heart defects in dogs. Journal of Small
Animal Practice 30, 153-165.
Rauch, R., Rauch, A., Koch, A., Zink, S., Kaulitz, R., Girisch, M., Singer, H., Hofbeck,
M., 2004. Laterality of the aortic arch and anomalies of the subclavian artery –
reliable indicators for 22q11.2 deletion syndromes? European Journal of
Paediatrics 163, 642-645.
Stoller, J.Z., Epstein, J.A., 2005. Identification of a novel nuclear localization signal in
TBX1 that is deleted in DiGeorge syndrome patients harbouring the 1223delC
mutation. Human Molecular Genetics 14, 885-892.
VanGundy, T., 1989. Vascular ring anomalies. Compendium on Continuing
Education for the Practicing Veterinarian 2, 36-48.
Yagi, H., Furutani, Y., Hamada, H., Sasaki, T., Asakawa, S., 2003. Role of TBX1 in
human del22q11.2 syndrome. Lancet 362, 1366-1373.
Werner, P., Raducha, M.G., Prociuk, U., Budarf, M., Henthorn, P.S., Patterson, D.F.,
1999. Comparative mapping of the DiGeorge region in the dog and exclusion of
linkage to inherited canine conotruncal heart defects. The Journal of Heredity 90,
494-498.
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
19
Wrede, J., Schmidt, T. 2003. OPTI-MATE Version 3.81. A management programme
useful to minimize inbreeding in endangered populations. Programme Manual.
Institute for Animal Breeding and Genetics, University of Veterinary Medicine
Hannover, Germany.
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
20
2.10 Appendix
Table 1: Comparison of coefficients of inbreeding among PRAA affected and a
contemporary group of German Pinscher as well as and relationship coefficients
among parents with PRAA affected progeny and a contemporary group of German
Pinschers
Parameter Contemporary group PRAA affected or German
pinschers with PRAA affected
offspring
P value
Mean coefficient of
relationship (%) 7.44 ± 7.45 11.04 ± 10.14 0.088
Mean coefficient of
inbreeding (%) 3.56 ± 2.98 6.76 ± 4.86 0.005
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
21
Fig. 1: Contrast oesophagram of puppy 2 (ventro-dorsal projection): Highly dilated
oesophagus (A) cranial to heart base.
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
22
Fig. 2 + 3: Findings in the autopsy of puppy 2. The oesophagus (A) is highly dilated
cranial to the heart. The left subclavian artery (B) originated in the distal right aortic
arch (C), coursed dorsal to the oesophagus and crossed over to the left side. The
ligamentum arteriosum (D) joins the pulmonary artery with the aberrant subclavian
artery.
Figure 2:
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
23
Fig. 2 + 3: Findings in the autopsy of puppy 2. The oesophagus (A) is highly dilated
cranial to the heart. The left subclavian artery (B) originated in the distal right aortic
arch (C), coursed dorsal to the oesophagus and crossed over to the left side. The
ligamentum arteriosum (D) joins the pulmonary artery with the aberrant subclavian
artery.
Figure 3:
Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German Pinschers
24
Fig. 4: Pedigree showing the German Pinschers affected by PRAA. In puppies 1-3
the specific form of PRAA with an aberrant left subclavian artery with a ligamentum
arteriosum joining the left subclavian artery and the main pulmonary artery was
diagnosed.
CHAPTER 3
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
Julia Menzel, Ute Philipp, Ottmar Distl
Institute for Animal Breeding and Genetics, University of Veterinary Medicine
Hannover, Foundation, Bünteweg 17p, 30559 Hannover
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
26
3 Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher 3.1 Abstract Persistent right aortic arch (PRAA) is a congenital vascular ring anomaly and
common in several dog breeds. In the German Pinscher, a rare form of this disease
occurs in which the persistent right aortic arch is associated with an aberrant left
subclavian artery and a ligamentum arteriosum originating at the aberrant left
subclavian artery (PRAA-SA-LA). In the present study, we analyzed the canine t-box
gene TBX1 for association with PRAA-SA-LA in the German Pinscher. We
genotyped 37 microsatellite markers on canine chromosome 26 (CFA26) in two
German Pinscher families and tested them for linkage and association. We found a
genome-wide significant genomic region on CFA26 which co-segregates with the
PRAA-phenotype in the German Pinschers. We also sequenced the whole genomic
sequence of the candidate gene TBX1 on CFA26. In addition, we sequenced partly
14 other genes located within the canine DiGeorge critical region (DGCR). The
search for single nucleotide polymorphisms (SNPs) within these genes revealed a
total of 23 polymorphisms. Two of these SNPs located within the canine TBX1 gene
were found to be associated with the PRAA-phenotype in the German Pinscher.
Additionally, we found 13 interbred SNPs in the TBX1 gene. All SNPs were located in
intronic regions.
3.2 Introduction Persistent right aortic arch (PRAA) represents the most common vascular ring
anomaly in dogs (Buchanan, 2004). Abnormal blood vessels and associated
structures form a complete circle around the trachea and the oesophagus. This
anomaly leads to a compression of the encircled structures and often causes
regurgitation in young dogs. In the German Pinscher, a rare form of PRAA occurs
which is associated with an aberrant left subclavian artery and a ligamentum
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
27
arteriosum that extended from the main pulmonary artery to the aberrant subclavian
artery instead of the aortic arch (Menzel and Distl, 2010). This additional anomaly
enforces the compression of the oesophagus and has only been reported in two
cases of other dog breeds before (House et al., 2005). Epidemiologic studies as well
as breeding studies have shown that German Shepherds, Irish Setters, Greyhounds
and German Pinschers are genetically predisposed to the development of PRAA
(Patterson, 1968; Patterson, 1989; Gunby et al., 2004; Menzel and Distl, 2010).
Comparative genetic research revealed that conotruncal heart defects in humans
have been associated with deletion of the chromosome 22q11.2 region which is also
known as the DiGeorge critical region (DGCR) (Rauch et al., 2004; Lee et al., 2006).
Monosomy 22q11.2 was found to be strongly associated with the presence of
subclavian artery anomalies (SAAs) (Rauch et al., 2004). The
DiGeorge/velocardiofascial syndrome (DGS/VCFS) is a relatively common human
disorder characterized by a wide range of developmental anomalies including
cardiovascular defects and defects of glands and facial structures. Investigations of
the potential role of one candidate gene for the DGS, TBX1, revealed that mice
heterozygous for a targeted mutation in the TBX1 gene had a high incidence of
cardiac outflow tract anomalies, one of the major abnormalities of the human
syndrome (Lindsay et al., 2001; Jerome and Papaioannou, 2001).
The TBX1 gene is a member of the t-box gene family of DNA binding transcription
factors. T-box genes have been shown to play an important role in the regulation of
developmental processes in humans and animals including angiogenesis, artery
morphogenesis, blood vessel development and remodelling, determination of left and
right symmetry, heart development and morphogenesis and mesoderm development
(Papaioannou and Silver, 1998; Smith, 1999). Haploinsufficiency of two other t-box
genes, TBX3 and TBX5, are associated with the human genetic diseases ulnar-
mammary syndrome and Holt-Oram syndrome (Bamshad et al., 1997; Basson et al.,
1997). The TBX1 gene is conserved in human, chimpanzee, mouse, rat, zebrafish
and Caenorhabditis elegans. A study in which comparative mapping of the DGCR in
the dog has been performed revealed that this region is mapped to the telomeric end
of chromosome 26 and appeared to be conserved in the dog (Werner et al., 1999).
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
28
In the present study, we evaluated the whole genomic structure of the canine TBX1
gene and 13 other genes located within the DGCR conserved in the dog and
screened exons with their flanking intronic regions for polymorphisms to be used for
linkage and association tests with the PRAA-phenotype in the German Pinscher.
3.3 Material and Methods
Animals, phenotypic data and DNA specimens
The Pinscher-Schnauzer-Klub e.V. (PSK) supplied pedigree data and we identified
pedigrees with PRAA-affected dogs. For the present analysis, we chose 45 dogs
from two different German Pinscher families. Altogether this study included 3 PRAA-
affected German Pinschers with the same rare form of PRAA (PRAA-SA-LA). After
first results of the linkage and association analyses (Table 1), we decided to classify
all available full-sibs of these affected puppies (n=10) as genetic carriers to reach
more reliable results. We also tested three unaffected dogs from other breeds as
control animals.
Two millilitres of EDTA blood (BIOTA) was obtained from each dog and DNA was
extracted using QIAamp 96 DNA Blood kit (Qiagen, Hilden, Germany).
Genotyping of microsatellites
We genotyped 37 microsatellite markers on canine chromosome 26 (CFA26) starting
at 11.90 Mb up to 37.90 Mb. Microsatellites were obtained by searching the Pubmed
database (dog genome assembly 2.1) (http://www.ncbi.nlm.nuh.gov/
entrez/query.fcgi) for known microsatellites with a distance of about 0.5 Mb to each
other. PCR primers are shown in Table 2. The PCR for genotyping of the
microsatellites started at 94°C for 4 min, followed by 38 cycles at 94°C for 30 sec,
optimum annealing temperature for 1 min, 72°C for 30 sec, and at 4°C for 10 min. All
PCR reactions were performed in 11.5-µl reactions using 6 pmol of each primer, 0.2
µl dNTPs (100 µM) and 0.1 µl Taq-DNA-Polymerase (5 U/µl) (Q-Biogen, Heidelberg,
Germany) in the reaction buffer supplied by the manufacturer for 2 µl template DNA.
The forward primers were labelled fluorescently with IRD700 or IRD800. For the
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
29
analysis of the marker genotypes, PCR products were size-fractionated by gel
electrophoresis on an automated sequencer (LI-COR, Lincoln, NE, USA) using 4%
polyacrylamide denaturing gels (Rotiphorese Gel40, Carl Roth, Karlsruhe). Allele
sizes were detected using an IRD700- and IRD800-labeled DNA ladder; the
genotypes were assigned by visual examination.
Non-parametric linkage analysis
A non-parametric multipoint linkage analysis was employed for the two German
Pinscher families using the MERLIN 1.1.2 software (Abecasis et al. 2002). This
analysis is based on allele sharing among affected individuals by identical-by-
descent methods (Kong and Cox 1997). Haplotypes were estimated using MERLIN
1.1.2 with the option “best”. A case-control analysis based on χ2-tests for genotypes,
alleles and trend of the most prevalent allele was also performed for the German
Pinschers. The CASECONTROL and ALLELE procedures of SAS/Genetics were
used for association tests, tests for Hardy-Weinberg equilibrium of genotype
frequencies and the estimation of allele frequencies (SAS Institute, 2005).
Structural and mutation analysis of TBX1 and 13 other genes
The dog-expressed sequence tag (EST) archive
(http://www.ncbi.nlm.nih.gov/genome/seq/CfaBlast.html) was searched for ESTs by
cross-species BLAST searches with the corresponding human reference mRNA
sequence for TBX1 (NM_080647.1). We found two canine ESTs (DN269432.1 and
DN_399703.1) isolated from lymph node and aorta tissue with 89% and 92% identity
to the human TBX1 mRNA sequence. A significant match to these canine ESTs was
found on canine chromosome 26 by means of BLASTN searches of the canine ESTs
against the dog genome assembly (Dog genome assembly 2.1). The genomic
structure of the canine TBX1 gene was determined with the Spidey mRNA-to-
genomic alignment program (http://www.ncbi.nlm.nih.gov/IEB/Research/
Ostell/Spidey/index.html).
For evaluation of TBX1 as candidate gene for PRAA in the German Pinscher, we
sequenced the whole genomic sequence of the canine TBX1 gene including more
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
30
than 400 bp upstream of the start codon and 3 kb downstream of the stop codon for
3 affected and 8 unaffected German Pinschers out of the two families mentioned
above. In addition, we sequenced partly the exons with flanking intronic regions of 13
other genes located within the DGCR conserved in the dog. All genes were located
on the telomeric end of canine chromosome 26 (CFA26), where the DGCR in the dog
is mapped to.
We used the genomic sequences of the canine genes (TBX1, ARVCV, COMT,
TXNRD2, GNBL1, SEP5, CDC45L, UFD1, MRPL40, HIRA, CLTCL1, SLC25A1,
GSC2 and DGS14) of the current dog genome assembly (dog genome assembly 2.1)
together with the mRNA sequences of human TBX1 gene to localize the exon/intron
boundaries of these genes in the dog. PCR primers were designed using the Primer3
program (http://frodo.wi.mit.edu/cgi-bin/primer3_www.cgi) based on the genomic
sequence for canine TBX1 (LOC60821) and the other 13 genes. The PCR primers
for the amplification of the genomic sequences are listed in Table 3. Sequence data
were analysed with Sequencher version 4.7 (GeneCodes, Ann Arboer, MI, USA).
All PCRs were performed in 50-µl reactions using 20 pmol of each primer, 40 µM
dNTPs, 0.5 U PeqLab-DNA-Polymerase (PeqLab, Erlangen, Germany) in the
reaction buffer supplied by the manufacturer, 5x PCR Enhancer 1 (PeqLab,
Erlangen, Germany), and 5% DMSO for 3 µl template DNA. The PCR conditions
were: 95°C for 5 min followed by 38 cycles of 95 °C for 30 s, optimum annealing
temperature for 30 s, 72°C for optimum elongation time, and 4°C for 10 min. All PCR
products were cleaned using the Nucleo-Fast PCR purification kit (Macherey-Nagel)
and directly sequenced with the DYEnamic ET Terminator kit (GE healthcare,
München, Germany) and a MegaBACE 1000 capillary sequencer (GE Healthcare).
3.4 Results and Discussion
Non-parametric linkage analysis
Table 4 shows the results of the non-parametric linkage analysis for all 37
microsatellite markers on CFA26 after classification of full sibs of affected animals as
genetic carriers. The highest and the only significant LOD scores of 0.93 and 0.81
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
31
were obtained for the markers 26_31.48 and 26_31.99 which are located in a
distance of 0.5 to 1 Mb to the candidate gene TBX1. The maximum achievable
Zmean was 2.47 indicating that the power of the analysis was high enough to detect
significant linkage. The error probabilities for linked markers ranged from 0.013 to
0.03. The polymorphism information content of the individual markers was between
48 and 73%.
Structural and mutation analysis of TBX1 and 13 other genes
The canine ESTs for TBX1 (DN269432.1 and DN399703.1), which were found by
cross-species BLAST searches with the corresponding human reference mRNA
sequences, mapped to the same position as the annotated gene for TBX1
(LOC608214). We performed a mutation analysis for the TBX1 gene due to the
significant linkage of the markers nearby. The canine TBX1 gene (LOC) consists of
seven exons interrupted by six introns. We sequenced the whole coding sequence of
the canine TBX1 gene. In addition, we sequenced 13 other genes partly which were
located in the former canine DiGeorge region: ARVCV, COMT, TXNRD2, GNB1L,
TBX1, SEP5, CDC45L, UFD1, MRPL40, HIRA, CLTCL1, SLC25A1, GSC2 and
DGS14. The search for sequence variations within these 14 genes revealed a total of
18 SNPs. Of these 18 SNPs, four were located in the intronic sequence of TBX1
while the other were located in the intronic sequences of GNBL1, TXNRD2, CDC45L,
UFD1, MRPL40, CLTCL1, SLC25A1 and GSC2. Additionally, we found 13 interbred
SNPs in the German Pinscher for the TBX1 gene which were all located in intronic
regions (Table 6).
Table 5 shows the results of the non-parametric linkage analysis for all these 18
SNPs. The χ2 –test statistics for distributions of genotypes between cases and
controls ranged from 0.24 to 10.00 and their error probabilities from 0.80 to 0.006.
The highest and only significant χ2 of 10.00 and 6.87 with error probabilities of 0.006
and 0.03 were detected for LOC608214:g.537G>A and LOC608214:g.4714C>T.
Therefore, it is very likely that the candidate gene TBX1 is involved in the
pathogenesis of PRAA in the German Pinscher. Sequencing of the TBX1 gene of
more affected dogs should be performed in order to confirm these results.
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
32
3.5 Acknowledgements
The authors would like to thank the Pinscher-Schnauzer-Klub e.V. (PSK) for
providing the data and the blood samples.
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
33
3.6 References
Abecasis GR, Cherny SS, Cookson WO, Cardon LR. Merlin-rapid analysis of dense
genetic maps using sparse gene flow trees. Nature Genetics 2002; 30: 97-101.
Bamshad, M., Lin, R.C., Law, D.J., Watkins, W.J., Krakowiak, P.A., Moore, M.W.,
Francescjini, P., Lala, R., Holmes, L.B., Gebuhr, T.C., Bruneau, B.G., Schinzel,
A., Seidmann, J.G., Seidmann, C.E., Jorde, L.B., 1997. Mutations in human
TBX3 alter limb, aprocine and genital development in ulnar-mammary syndrome.
Nature Genetics 15, 21-29.
Basson, C.T., Bachinsky, D.R., Lin, R.C., Levi, T., Elkins, J.A., Soults, J., Grayzel, D.,
Kroumpuzou, E., Traill, T.A.,Leblanc-Straceski, J., Renault, B., Kucherlapati, R.,
Seidmann, J.G., Seidmann, C.E., 1997. Mutations in human cause limb and
cardiac malformation in Holt-oram syndrome. Nature Genetics 15, 30-35.
Buchanan, J.W., 2004. Tracheal signs and associated vascular anomalies in dogs
with persistent right aortic arch. Journal of Veterinary Internal Medicine 18, 510-
514.
Clark LA, Tsai KL, Steiner JM, Williams DA, Guerra T, Ostrander EA, Galibert F,
Murphy KE. Chromosome-specific microsatellite multiplex sets for linkage studies
in the domestic dog. Genomics 2004; 84: 550–554.
Gunby, J.M., Hardie, R.J., Bjorling, D.E., 2004. Investigation of the potential
heritability of persistent right aortic arch in Greyhounds. Journal of the American
Veterinary Medical Association 224, 1120-1121.
Guyon R, Lorentzen TD, Hitte C, Kim L, Cadieu E, Parker HG, Quignon P, Lowe JK,
Renier C, Gelfenbeyn B, et al. A 1-Mb resolution radiation hybrid map of the
canine genome. Proceedings of the National Academy of Sciences of the United
States of America 2003; 100: 5296–5301.
House, A.K., Summerfield, N.J., German, A.J., Noble, P.J.M., Ibbarola, P.,
Brockmann D.J., 2005. Unusual vascular ring anomaly associated with a
persistent right aortic arch in two dogs, Journal of Small Animal Practice 6, 585-
590.
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
34
Jerome, L.A., Papaioannou, V. E., 2001. DiGeorge syndrome phenotype in mice
mutant for the T-box gene TBX1. Nature Genetics 27, 286-291.
Kong A, Cox NJ. Allele-sharing models: LOD scores and accurate linkage tests. The
American Journal of Human Genetics 1997; 61: 1179-1188.
Lee, M.-L., Chen, H.-N., Chen, M., Tsao, L.-Y., Wang, B.-T., Lee, M.-H., Chiu, I.-S.,
2006. Persistent fifth aortic arch associated with 22q11.2 deletion syndrome.
Journal of the Formosan Medical Association 105, 284-289.
Lindsay, E.A., Vitelli, F., Su, H., Morishima, M., Huynh, T., 2001. Tbx1
haploinsufficiency in the DiGeorge syndrome region causes aortic arch defects in
mice. Nature 410, 97-101.
Menzel, J, Distl, O., 2010. Unusual subclavian artery associated with a persistent
right aortic arch and an aberrant left subclavian artery in German Pinschers. The
Veterinary Journal, Epub ahead of print.
Papaioannou, V.E., Silver, L.M., 1998. The T-box gene family. Bioessays 20, 9-19.
Patterson, D.F., 1968. Epidemiologic and Genetic Studies of Congenital Heart
Disease in the dog. Circulation Research 23, 171-2002.
Patterson, D.F., 1989. Hereditary congenital heart defects in dogs. Journal of Small
Animal Practice 30, 153-165.
Rauch, R., Rauch, A., Koch, A., Zink, S., Kaulitz, R., Girisch, M., Singer, H., Hofbeck,
M., 2004. Laterality of the aortic arch and anomalies of the subclavian artery –
reliable indicators for 22q11.2 deletion syndromes? European Journal of
Paediatrics 163, 642-645.
SAS Institute. SAS/Genetics, Version 9.1.3. Cary, NC, USA, 2005
Smith, J., 1999. T-box genes. What they do and how they do it. Trends in Genetic 15,
154-158.
Werner, P., Raducha, M.G., Prociuk, U., Budarf, M., Henthorn, P.S., Patterson, D.F.,
1999. Comparative mapping of the DiGeorge region in the dog and exclusion of
linkage to inherited canine conotruncal heart defects. The Journal of Heredity 90,
494-498.
3.7 Appendix Table 1: Non-parametric test statistics Zmean and LOD Score, their error probabilities (PZ, PL), polymorphism information
content (PIC), χ2-tests for allele and genotype distribution of the case-control analysis, degrees of freedom (DF) and their
corresponding error probabilities (P) for the microsatellite markers on canine chromosome 26 (CFA26) in the German
Pinscher (full-sibs were not classified as genetic carriers)
Test for linkage Test for association Marker Position
on
CFA26
PIC (%)
Zmean PZ LOD
score
PL χ2 geno-
type
DF P geno-
type
χ2
allele
DF P
allele
DTR26.9 11.90 0.37 -0.99 0.8 -0.23 0.8 7.84 3 0.04 5.59 2 0.06
26_20.49 20.49 0.20 -1.40 0.9 -0.30 0.9 0.16 1 0.68 0.13 1 0.71
26_21.14 21.14 0.75 -1.41 0.9 -0.30 0.9 24.22 14 0.04 19.80 6 0.002
26_21.69 21.69 0.66 -0.91 0.8 -0.22 0.8 17.09 11 0.10 2.53 5 0.77
REN131L06 22.30 0.54 -0.71 0.8 -0.18 0.8 10.90 5 0.05 3.40 2 0.18
26_22.49 22.49 0.67 0.70 0.2 0.17 0.2 27.61 9 0.001 14.53 5 0.1
26_22.92 22.92 0.73 -0.87 0.8 -0.21 0.8 25.98 18 0.10 22.43 9 0.007
REN01O23 23.40 0.29 -1.26 0.9 -0.28 0.9 0.70 2 0.70 0.19 2 0.66
CRYBB2_26_23.50 23.50 0.58 -1.37 0.9 -0.29 0.9 12.44 7 0.08 4.34 7 0.22
26_23.54 23.54 0.46 -1.41 0.9 -0.30 0.9 3.58 5 0.60 3.68 2 0.15
CRYBB_26_23.59 23.59 0.61 -1.33 0.9 -0.29 0.9 13.62 9 0.13 5.63 4 0.22
26_24.11 24.11 0.68 -0.62 0.7 -0.16 0.8 6.22 6 0.39 8.96 5 0.11
26_24.49 24.49 0.32 -0.38 0.6 -0.10 0.8 8.31 3 0.03 5.20 2 0.07
26_25.09 25.09 0.75 0.00 0.5 0.00 0.5 18.05 12 0.11 7.60 5 0.17
Evaluation of the canine TB
X1 gene as candidate for a rare fom
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26_26.06 26.06 0.62 -0.66 0.7 -0.17 0.8 26.58 5 0.005 7.39 5 0.19
DGN10 26.10 0.75 -0.68 0.8 -0.17 0.8 26.43 14 0.02 17.41 9 0.04
26_26.46 26.46 0.36 -0.60 0.7 -0.15 0.8 8.19 3 0.04 5.69 2 0.05
26_26.98 26.98 0.37 -0.50 0.7 -0.13 0.8 2.30 2 0.31 0.44 1 0.50
26_28.01 28.01 0.54 -0.39 0.7 -0.11 0.8 9.94 7 0.19 11.13 3 0.01
26_29.04 29.04 0.04 -0.27 0.6 -0.08 0.7 0.87 1 0.34 0.85 1 0.35
26_29.57 29.57 0.59 -0.21 0.6 -0.06 0.7 15.57 7 0.02 7.53 3 0.05
26_30.00 30.00 0.59 -0.17 0.6 -0.05 0.7 25.24 8 0.001 19.67 4 0.0005
26_30.92 30.92 0.58 -0.06 0.5 -0.02 0.6 14.50 7 0.04 9.75 3 0.02
26_31.48 31.48 0.73 -0.00 0.5 -0.00 0.5 29.18 16 0.02 26.88 8 0.0007
26_31.99 31.99 0.48 0.15 0.4 0.04 0.3 15.52 4 0.003 11.07 3 0.01
REN88N03 32.67 0.58 0.79 0.2 0.19 0.2 20.78 6 0.002 14.08 3 0.002
26_32.70 32.70 0.35 0.94 0.2 0.22 0.2 11.61 6 0.07 12.30 4 0.01
26_32.84 32.84 0.27 1.18 0.12 0.26 0.14 1.22 2 0.54 1.09 2 0.57
26_33.03 33.03 0.44 1.34 0.09 0.29 0.12 8.57 4 0.07 7.58 3 0.05
26_33.10 33.10 0.67 1.41 0.08 0.30 0.12 22.65 11 0.01 16.67 5 0.005
REN276I22 33.40 0.37 1.39 0.08 0.30 0.12 3.95 2 0.13 1.20 1 0.27
26_33.44 33.44 0.04 1.39 0.08 0.30 0.12 0.87 1 0.34 0.85 1 0.35
26_33.95 33.95 0.51 1.36 0.09 0.29 0.12 16.99 7 0.01 12.81 3 0.005
26_34.51 34.51 0.31 1.33 0.09 0.29 0.12 2.94 4 0.56 2.80 3 0.42
26_35.13 35.13 0.64 1.30 0.10 0.28 0.13 27.47 8 0.0005 12.92 3 0.004
FH2130 35.50 0.58 1.28 0.10 0.28 0.13 8.20 7 0.31 3.06 4 0.54
CA26.733 37.90 0.23 1.16 0.12 0.26 0.14 6.91 3 0.07 6.98 2 0.03
Evaluation of the canine TB
X1 gene as candidate for a rare form
of persistent right aortic arch in the G
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Table 2: PCR primers, their position, product size and annealing temperature (Ta) for the amplification of microsatellite
markers on canine chromosome 26 (CFA26)
Primer Sequence (5’ – 3’) of primers Position on CFA26 (Mb) Ta (°C) Product size (bp) DTR26.9_F TAAGCACTAAAGTTTCCCCA 11.551630 – 1.551934 DTR26.9_R GATAAAGACCATTGTGAGCC
58 294 - 314
26_20.49_F CCTAAGCTAGACATTGCGCCC 20.492686 – 20.492734 26_20.49_R CCCGAATGACCTTGACAAAT
60 281
REN299M21_F AAAGTTGCCCACCTGTTGAC 20430405 - 20430646 REN299M21_R GACTTGGAGAGGACTGTGCC
60 242
26_21.14_F AATTCCTCATTCTGATTCTCCAC 21.148368 – 21.148443 26_21.14_R CGGATGTTATGATATGCAAATAAGC
60 187
26_21.69_F CAGCCAGAGGACAAACTCTATCTA 21.699561 – 21.699692 26_21.69_R GGGTTTGTATTCAAGAGCTCCA
60 230
REN131L06 GCTGTCCTGCACTTTTCCTC 23.083557 – 23.083675 REN131L06 GTTAAGGAATAGTTGGGGGTCC
54 117
26_22.49_F CTTAGGGCATTCCGTTACCA 22.491459 – 22.491547
26_22.49_R TAGCTCCTGGCACGATTCTT 60 222
26_22.92_F TTGGGGTCTGGAGTTGTCTC 22.928417 – 22.928553 26_22.92_R TGTGTCCTGTGACTCCCAAA
60 319
REN01O23_F TTCCCTGCAGCCCTTCCTCA 32.676461 – 32.676611 REN01O23_R TGTGCCTCATTCCTTTTTAT
60 149 - 167
Evaluation of the canine TB
X1 gene as candidate for a rare form
of persistent right aortic arch in the G
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CRYBB_26_23.50_F TACTGACAAGTGCAGTGGGG 23.503427 – 23.503755 CRYBB_26_23.50_R CGAAAAATGCCCTGATGAAT
55 334 - 350
26_23.54_F TGCATACCCTCCCAACTTTC 23.542350 – 23.542409 26_23.54_R TTCAGGAAGCACAACTTATACAATG
60 287
CRYBB_23.59_F TCTGCCTATGTCTCTGCCTCTC 23.591923 – 23.592215 CRYBB_23.59_F GGCACCTATTCTGGGAAACTTT
55 286 - 294
26_24.11_F AAGTCTTAACATCCTCTTAGCACCT 24.115260 – 24.115323
26_24.11_R GAGAGTCGGGCTTCTGGTC
58 319
26_24.49_F AAGGCATGTAACAAGGCACC 24.495412 – 24.495508 26_24.49_R TCTGAAATTCCCTGGCACTC
60 272
26_25.09_F TGCAGCATGGATACAGTAAAGAA 25.099832 – 25.099892
26_25.09_R ACTTACCCAGACCACTCAGCTC
60 317
26_26.06_F GGCTTGGAATGGAACTGAGA 26.066507 – 26.066602 59 284 26_26.06_R GAAGTGGTTGAAGGGTACCAA DGN10_F TCTGCCTATGTCTCTGCCTCTC 26.049203 – 26.049455 62 243-267 DGN10_R GGCACCTATTCTGGGAAACTTT 26_26.46_F TACAGCCACATGGCTCAAAA 26.461766 – 26.461799 60 221 26_26.46_R CTCTTTCCTGCATTTCTGCC 26_26.98_F TACTGACAAGTGCAGTGGGG 26.980932 – 26.980965 60 225 26_26.98_R CGAAAAATGCCCTGATGAAT 26_28.01_F GGCATACATTCTACAAAGCAGGA 28.014576 – 28.014623 60 206 26_28.01_R ATGCAGAAGTCCGCCAATAG 26_29.04_F AGGGAGAAGGGTCATCTTGC 29.043396 – 29.043430 60 183 26_29.04_R TGGAAGGATGGAGAATCCTG
38
Evaluation of the canine TB
X1 gene as candidate for a rare form
of persistent right aortic arch in the G
erman P
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49
26_29.57_F AAAGAGGACACACATTGCCC 29.579833 – 29.579901 60 184 26_29.57_R TCAGGAACAGGATCTGGGAG 26_30.00_F GGGAGATATGCAAGAGGACAAG 30.003674 – 30.003740 59 238
26_30.00_R ATCCGGGAGGAAGATTCAGT
26_30.92_F GGGCCCCTGTGTGTATCTTA 30.923484 – 30.923515 60 285 26_30.92_R GGATCAGAAAATGGAGCTGG 26_31.48_F GCAGCAGTTTGAGAGGGGTA 31.480708 – 31.480779 60 280 26_31.48_R ACGGAAGGCACATTTTCTTG 26_31.99_F TCAGTATTTCTCAAACCCTGTTTTT 31.996804 – 31.996840 60 236 26_31.99_R CTCTAGGCCAGAAGGCATTG REN88NO3_F TGCTTCTTGACTTCCCCTACA 32676461-32676611 60 149-167 REN88NO3_R GGTTTGCCTCCCTCTATGC 26_32.70_F AGAGGCCCAGAGCCATAGTC 32.705104 – 32.705145 60 144
26_32.70_R TGACTGGGGAGACTTCCTTG
26_32.84_F GTGGGGTGAACACCTGAAAC 32.845059 – 32.845100 60 259
26_32.84_R TTGTCCTCAGCCACAAGATG
26_33.03_F TTAGGGCAGCAGTCACACAG 33.030089 – 33.030124 60 332
26_33.03_R TATCGACCTGCCTTTTCCAC
26_33.10_F AGTGGTTGGGCAATTTTCTG 33.102932 – 33.102995 60 231 26_33.10_R TCCCTGTCCTCCTCCTTTCT REN276I22_F AACAATGTCCACAACAGCCA 34554897-34555065 58 159-165 REN276I22_R CATTTTTGTGATGGCTGAACA 26_33.44_F ATTCACAGGCCACAGAGGTC 33.445256 – 33.445287 60 316
26_33.44_R CAAGGTCTACCGATGTGCAA
Evaluation of the canine TB
X1 gene as candidate for a rare fom
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49
26_33.95_F GTTGGGTGTGACATGACAGG 33.952079 – 33.952113 60 300 26_33.95_R AGATAAAGAGCCCCAGCCTC 26_34.51_F GGAGAGAGTGGTATGATTTGGG 34.511467 – 34.511540 60 178 26_34.51_R TTTGGTTTGCATATGGCTTG 26_35.13_F CCCGTGAAGATTTGGTGACT 35.133831 – 35.133908 60 332
26_35.13_R TTCGGTTACAAGGTTGTCAGATT
FH2130_F GCTGTCCTGCACTTTTCCTC 35.504365 – 35.504662 62 292 - 312 FH2130_R GTTAAGGAATAGTTGGGGGTCC CA26.733_F CCCTCTACTTATGTCTCGGCC 37.905674 – 35.905923 58 243 - 255 CA26.733_R GAGAGGAGAAACAACCAACACC
Evaluation of the canine TB
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of persistent right aortic arch in the G
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Table 3: PCR primers, their position, product size and annealing temperature (Ta) for the amplification of the genomic
sequence of genes in the candidate gene region on canine chromosome 26 (CFA26)
Gene symbol Primer Sequence (5’ – 3’) of primers Position on CFA26 (Mb) Ta (°C) Product size (bp) ARVCF ARVCF_2F GTTCGGGCACTCTCTGTG 32.417021 - 32.417476 57 456 ARVCF_2R TCTGATGAACAGGCAGGAC COMT COMT_1F GACGGAGCATCTCTAACTGC 32.428265 - 32.428754 57 490 COMT_1R AAAGCTCCTTTCTTTCATTCC TXNRD2 TXNRD2_1F CCCCACGACCTGTCTGTC 32.469446 - 32.469996 60 551 TXNRD2_1R GGTGTGGTTCTGAGGGTCTG GNB1L GNB1L_1F TTCTCCCACAAAGTGGCTTC 32.550052 - 32.550511 59 460 GNB1L_1R GTCACCAGGGACATGGAAG TBX1 TBX1_1F CTTCTTTCCCCACTTCTGTTC 32.602435 - 32.603160 58 726 TBX1_1R GTCACCAGGGACATGGAAG TBX1_2F TGTAGGGGTGGTGGTGCAG 32.595984 - 32.596557 63 574 TBX1_2R GCGCCGAGAAAGGTAGGG TBX1_3F GCTGGTATCTGTGCATGGAG 32.597535 - 32.598190 58 656 TBX1_3R ACAACCTGCTGGATGACAAC TBX1_4F ACACGAGACGACAGGAAGC 32.598526 - 32.599219 59 685 TBX1_4R CGACTACATGCTCCTCATGG TBX1_5F CCAGCAGGTTGTTGGTCAG 32.598179 - 32.598844 60 666 TBX1_5R TTTCTTCGCGTCTCCTTCTC TBX1_6F CAGCCTCGGTCTTAGGGCCTGT 32.602933 - 32.606481 68 3549 TBX1_6R GCCTGAGTCTAGCCGCTTGCAG TBX1_7F ACCGTCACCAGGGACATGGAAG 32.599850 - 32.603163 68 3314
41
Evaluation of the canine TB
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TBX1_7R AATCCCTCCAACGAGGCTGAGG TBX1_8F TACTGTGGCCGGTGGGAACTTG 32.596751 - 32.599961 68 3211 TBX1_8R GGTTCGCAGTCTCGGAAGCCTTT TBX1_9F GCCTCAAGCAGCCTCCTCTCCT 32.595410 - 32.597279 68 1870 TBX1_9R GCCGGCGCGGTATCTACAGTATC SEP5 SEP5_1F CAGCACTGAGCAGCTTCC 32.639219 - 32.639904 58 686 SEP5_1R AGGAGGGGTGAGTATTGAGG CDC45L CDC45L_1F GACAGTAAGGGAAATAAAAGTTAG 32.798401 - 32.798813 56 413 CDC45L_1R TTTGCAACTGTTTTGTGTTTG UFD1 UFD1_1F CCCAGTCAGCAGAGGTCAC 32.819104 - 32.819875 57 772 UFD1_1R AAAAGACTTATGTACCAGAGGAAC MRPL40 MRPL40_1F CCAACTCCCACAAGACAGAG 32.841114 - 32.841866 58 753 MRPL40_1R AAATCCTGSSSSGTCCACAG HIRA HIRA_1F GTGGACCTCCCACTATGATG 32.855094 - 32.855822 58 729 HIRA_1R TTGAGCATTTCTGGGTTCTC CLTCL1 CLTCL1_1F GTGCGTGTTACCCAGCTTAG 33.029143 - 33.029887 58 745 CLTCL1_1R GCACCGTTATTCATCAGAGG SLC25A1 SLC25A1_1F TGGAATCCTGAGAACCAGTG 33.0700779 - 33.070767 58 689 SLC25A1_1R CACATCCAGAGGAGTGTTCC GSC2 GSC2_1F CCGTGAGCCAACTGTGTC 33.098171 - 33.098779 57 610 GSC2_1R GCAAGTCTGGAGAAAACCAC DGS14 DGS14_1F TCTTTCCTGACGTGGAGAAG 33.119158 - 33.119945 58 788 DGS14_1R CATGCACAGAAGCAAGACAG
Evaluation of the canine TB
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Table 4: Non-parametric test statistics Zmean and LOD Score, their error probabilities (PZ, PL), polymorphism information
content (PIC), χ2-tests for allele and genotype distribution of the case-control analysis, degrees of freedom (DF) and their
corresponding error probabilities (P) for the microsatellite markers on canine chromosome 26 (CFA26) in the German
Pinscher (full-sibs were classified as genetic carriers)
Test for linkage Test for association Marker Position
on
CFA26
PIC (%)
Zmean PZ LOD
score
PL χ2 geno-
type
DF P geno-
type
χ2
allele
DF P
allele
DTR26.9 11.90 0.37 -0.72 0.8 -0-04 0.7 7.84 3 0.04 5.59 2 0.06
26_20.49 20.49 0.20 -0.77 0.8 -0.04 0.7 0.16 1 0.68 0.13 1 0.71
26_21.14 21.14 0.75 -0.77 0.8 -0.04 0.7 24.22 14 0.04 19.80 6 0.002
26_21.69 21.69 0.66 -0.19 0.6 -0.01 0.6 17.09 11 0.10 2.53 5 0.77
REN131L06 22.30 0.54 -0.48 0.7 -0.03 0.6 10.90 5 0.05 3.40 2 0.18
26_22.49 22.49 0.67 -0.14 0.6 -0.01 0.6 27.61 9 0.001 14.53 5 0.1
26_22.92 22.92 0.73 -0.16 0.6 -0.01 0.6 25.98 18 0.10 22.43 9 0.007
REN01O23 23.40 0.29 0.03 0.5 0.00 0.5 0.70 2 0.70 0.19 2 0.66
CRYBB2_26_23.50 23.50 0.58 0.05 0.5 0.00 0.5 12.44 7 0.08 4.34 7 0.22
26_23.54 23.54 0.46 0.08 0.5 0.01 0.4 3.58 5 0.60 3.68 2 0.15
CRYBB_26_23.59 23.59 0.61 0.12 0.5 0.01 0.4 13.62 9 0.13 5.63 4 0.22
26_24.11 24.11 0.68 0.52 0.3 0.28 0.13 6.22 6 0.39 8.96 5 0.11
26_24.49 24.49 0.32 -0.13 0.6 -0.01 0.6 8.31 3 0.03 5.20 2 0.07
26_25.09 25.09 0.75 -0.77 0.8 -0.04 0.7 18.05 12 0.11 7.60 5 0.17
26_26.06 26.06 0.62 1.15 0.12 0.23 0.2 26.58 5 0.005 7.39 5 0.19
DGN10 26.10 0.75 1.35 0.09 0.26 0.14 26.43 14 0.02 17.41 9 0.04
43
Evaluation of the canine TB
X1 gene as candidate for a rare form
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26_26.46 26.46 0.36 1.41 0.08 0.27 0.13 8.19 3 0.04 5.69 2 0.05
26_26.98 26.98 0.37 1.47 0.07 0.28 0.13 2.30 2 0.31 0.44 1 0.50
26_28.01 28.01 0.54 1.24 0.11 0.24 0.15 9.94 7 0.19 11.13 3 0.01
26_29.04 29.04 0.04 0.97 0.2 0.18 0.2 0.87 1 0.34 0.85 1 0.35
26_29.57 29.57 0.59 0.81 0.2 0.15 0.2 15.57 7 0.02 7.53 3 0.05
26_30.00 30.00 0.59 0.67 0.3 0.12 0.2 25.24 8 0.001 19.67 4 0.0005
26_30.92 30.92 0.58 0.33 0.4 0.05 0.3 14.50 7 0.04 9.75 3 0.02
26_31.48 31.48 0.73 2.47 0.007 0.93 0.02 29.18 16 0.02 26.88 8 0.0007
26_31.99 31.99 0.48 2.23 0.013 0.81 0.03 15.52 4 0.003 11.07 3 0.01
REN88N03 32.67 0.58 1.82 0.03 0.49 0.07 20.78 6 0.002 14.08 3 0.002
26_32.70 32.70 0.35 1.20 0.12 0.34 0.10 11.61 6 0.07 12.30 4 0.01
26_32.84 32.84 0.27 -0.63 0.7 -0.03 0.7 1.22 2 0.54 1.09 2 0.57
26_33.03 33.03 0.44 -0.57 0.7 -0.03 0.6 8.57 4 0.07 7.58 3 0.05
26_33.10 33.10 0.67 -0.70 0.8 -0.04 0.7 22.65 11 0.01 16.67 5 0.005
REN276I22 33.40 0.37 -0.81 0.8 -0.04 0.7 3.95 2 0.13 1.20 1 0.27
26_33.44 33.44 0.04 -0.81 0.8 -0.04 0.7 0.87 1 0.34 0.85 1 0.35
26_33.95 33.95 0.51 -0.80 0.8 -0.04 0.7 16.99 7 0.01 12.81 3 0.005
26_34.51 34.51 0.31 -0.79 0.8 -0.04 0.7 2.94 4 0.56 2.80 3 0.42
26_35.13 35.13 0.64 -0.77 0.8 -0.04 0.7 27.47 8 0.0005 12.92 3 0.004
FH2130 35.50 0.58 -0.76 0.8 -0.04 0.7 8.20 7 0.31 3.06 4 0.54
CA26.733 37.90 0.23 -0.70 0.8 -0.04 0.7 6.91 3 0.07 6.98 2 0.03
Evaluation of the canine TB
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of persistent right aortic arch in the G
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Table 5: Heterozygosity (HET), polymorphism information content (PIC) and χ2-tests of the case-control analysis with their
corresponding error probabilities (P) for the single nucleotide polymorphisms (SNPs) in the canine DiGeorge region on
canine chromosome 26 (CFA26) in German Pinschers
Gene Polymorphism PIC (%) HET (%) χ2 genotype P genotype χ2 allele P allele GNB1L g.29575C>T 0.28 0.45 0.74 0.38 0.52 0.46 (LOC486416) g.29586C>? 0.31 0.54 0.43 0.06 2.14 0.14 TBX1 g.537G>A 0.37 0.70 10.00 0.006 0.47 0.49 (LOC608214) g.776T>C 0.35 0.30 1.90 0.38 0.01 0.91 g.907A>G g.3447T>C g.3910T>G g.3925T>C g.4714C>T 0.37 0.70 6.87 0.03 0.05 0.82 g.4986C>T g.5426G>T 0.37 0.50 1.11 0.57 0.47 0.49 TXNRD2 g.19585T>C 0.31 0.54 0.24 0.62 0.15 0.69 (LOC608155) g.19722G>C 0.31 0.54 0.24 0.62 0.15 0.69 CDC45L g.6758G>A 0.35 0.54 0.49 0.78 0.03 0.85 (LOC486421) UFD1 g.18930C>T 0.36 0.63 0.43 0.80 0.19 0.65 (LOC608300) g.14120T>C 0.31 0.54 0.74 0.38 0.46 0.49 g.13995A>C 0.36 0.63 0.43 0.80 0.19 0.65 g.13906C>T 0.36 0.63 0.43 0.80 0.19 0.65 MRPL40 g.1855T>C 0.20 0.27 3.22 0.07 2.71 0.09 (LOC477567)
Evaluation of the canine TB
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49
SLC25A1 g.2206T>C 0.15 0.18 0.91 0.33 0.82 0.36 (LOC608348) GSC2 g.1242A>G 0.33 0.45 0.91 0.63 0.87 0.35 (LOC608374) g.754T>C 0.31 0.36 0.49 0.78 0.46 0.49
CLTCL1 g.55944C>A 0.36 0.63 2.35 0.30 0.28 0.59 (LOC477568)
46
Evaluation of the canine TB
X1 gene as candidate for a rare form
of persistent right aortic arch in the G
erman P
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Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
47
Table 6: Interbred single nucleotide polymorphisms (SNPs) in the German Pinscher for the canine TBX1 gene SNP LOC608214 Location g.556A>G 5’ UTR g.938InG 5’ UTR g.3115DelC Intron 1 g.3122InG Intron 1 g.3546A>G Intron 1 g.3586A>T Intron 1 g.3587A>G Intron 1 g.3588A>G Intron 1 g.3846T>C Intron 1 g.3847T>C Intron 1 g.6376G>T Intron 3 g.6744InC Intron 4 g.6799DelT Intron 4
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
48
Figure 1: Shown are the alignment of the canine TBX1 protein (325 amino acids) with
the known orthologous protein sequences derived from GenBank entries with the
accession nos. NP_542378 (human transcript variant C) and NP_035662 (mouse).
Residues identical to the dog are indicated by asterisks.
dog MDARSPLSP----------RASAFSIASLVAAEAA--------ERSARLG 32
human -MHF*TVTR----------DME**TAS**SSLG**GGFPGAASPGADPY* 39
mouse --MI*AV*SPWLTQLSHFCDVA**AAS**SGLGSP-------SPGADPF* 41
dog PR-------------------SSDPAKLRRLLGSPAGMHFSTVTRDMEGE 63
human **EPPPPPPRYDPCAAAAPGAPGP*PPPHAYPFA**AGAATSAAAEP**P 89
mouse **EPPPP--RYDPCAAVP-GAPGP*PP-RAYPFA**PGAAGSSAAES**P 87
dog PPCCVYGD--PPARPPATAAVGPRVTG----------G-----LAAPRRM 96
human GAS*AAAAKA*VKKNAKV*G*SVQLEMKALWDEFNQL*TEMIVTK*G*** 139
mouse GASRAAAVKA*VKKNPKV*S*SVQLEMKALWDEFNQL*TEMIVTK*G*** 137
dog FPTFQVKLFGMDPMADYMLLMDFVPVDDKRYRYAFHSSSWLVAGKADPAT 146
human ************************************************** 189
mouse ************************************************** 187
**************************************************
dog PGRVHYHPDSPAKGAQWMKQIVSFDKLKLTNNLLDDNGHIILNSMHRYQP 196
human ************************************************** 239
mouse ************************************************** 237
dog RFHVVYVDPRKDSEKYAEENFKTFVFEETRFTAVTAYQNHRITQLKIASN 246
human ************************************************** 289
mouse ************************************************** 287
Evaluation of the canine TBX1 gene as candidate for a rare form of persistent right aortic arch in the German Pinscher
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dog PFAKGFRDCDPEDWPRNHRPGALPLMSAFARSRNPWPPPRSPTAPRK--- 293
human ***********************************VAS*TQ*SGTE*DAA 339
mouse *************************V*********VAS*TQ*NGSD*DAA 337
dog -TRPRLGANSS--ATRADR-------------------------PCSGTR 315
human EA*REFQRDAGGP*VLG*PAHPPQLLARVLSPSLPGAGGAGGLV*LP*AP 389
mouse EA*REFDRDSG-P*ALG*ATHPPQLLARVLSPALP---GPGGLV*LP*GS 383
dog RTRRS------------------------------CWLGC---------- 325
human GG*P*PPNPELRLEAPGASEPLHHHPYKYPAAAYDHY**AKSRPAPYPLP 439
mouse GG*H*PPHADLRLEAPGASEPLHHHPYKYPAAAYDHY**AKSRPAPYPLP 433
dog --------------------------------------------------
human GLRGHGYHPHAHPHHHHHPVSPAAAAAAAAAAAAAAANMYSSAGAAPPGS 489
mouse GLRGHGYHPHAHPHAHPHHHHHPAVNPAAAAAAAAAANVYSSA-AAPPGA 482
dog ------
human YDYCPR 495
mouse YDYCPR 488
CHAPTER 4
Prevalence and formation of primary cataracts in the German Pinscher population in Germany
Julia Menzel, Ottmar Distl
Institute for Animal Breeding and Genetics, University of Veterinary Medicine
Hannover, Foundation, Bünteweg 17p, 30559 Hannover, Germany
Accepted for publication in: Veterinary Ophthalmology
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
52
3 Prevalence and formation of primary cataracts in the German Pinscher population in Germany
3.1 Abstract
Objective: The objective of this study is to describe the prevalence and formation of
primary non-congenital cataracts (CAT) in the German Pinscher population in
Germany and to analyze the familial occurrence and the mode of inheritance in this
dog breed.
Materials: Data included veterinary records for presumed inherited eye diseases of
German Pinschers born between 1993 and 2008 that were examined between 1997
and 2009 by certified veterinary ophthalmologists which were provided by the
German panel of the European Eye Scheme for diagnosis of inherited eye diseases
in animals (DOK). A total of 443 eye examinations reports of 261 dogs were
analyzed.
Results: CAT was diagnosed in 40 (15.33 %) of the examined dogs. The pedigree
included 58 ophthalmologically examined dogs with 20 unaffected and 38 affected
dogs. The coefficient of relationship as well as the coefficient of inbreeding was
significantly higher in the group of CAT-affected dogs than in a contemporary group
of German Pinschers. Simple segregation analysis revealed a recessive mode of
inheritance.
Conclusions: A bilateral anterior cortical CAT was the most prevalent CAT form
among close relatives in German Pinschers. The pedigrees supported a monogenic
autosomal recessive inheritance pattern. In order to improve breeding strategy
ophthalmologic examinations should be compulsory for all breeding animals. The
molecular genetic basis should be unravelled to avoid breeding with affected animals
and carriers so far.
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
53
3.2 Introduction
Primary non-congenital cataracts (CAT) are a leading cause of visual impairment and
blindness in purebred dogs. More than 120 dog breeds world wide are affected by
this eye disease, the reported prevalences range between 1.8 and 88.0%. Cataracts
in adult dogs have been shown to be hereditary in several dog breeds including
Leonbergers1, Entlebucher Mountain Dog2,3,4, Bichon Frise5,6, Tibetan Terriers7,
Chow Chows8, Golden and Labrador Retrievers9,10,11,12, German Shepherds13,
Standard Poodles14, Miniature Schnauzers15,16, West Highland White Terriers17,
Welsh Springer Spaniels18, Chesapeake Bay Retrievers19, Boston Terriers20,
Staffordshire Bull Terriers20, American Cocker Spaniels21,22, Cocker Spaniels23,
Afghan Hounds24, Old English Sheep Dogs25 and Beagles26. In the majority of the
dog breeds, a recessive mode of inheritance is assumed, but also dominant patterns
are described. In the German Pinscher breed in Finland, an autosomal recessive or
incomplete dominant inheritance pattern has been suspected27. CAT in German
Pinschers is only reported in one study based on eye examinations of 122 dogs of
the German Pinscher population in Finland27. The prevalence of CAT in this study
was 7.4% (n = 9) and the median age of manifestation was 9 years; the youngest
reported CAT case was a 4.7-year-old dog. Cataracts were posterior and
subcapsular in four dogs and in the anterior part of the lens in five dogs.
Because the only effective therapy known yet is the surgical intervention, the
containment of this inherited disease has large impact for animal welfare. In most
dog breeding associations, regular ophthalmologic examinations, carried out by
specialized ophthalmologists, are compulsory to achieve a breeding license in order
to reduce the risk of transmission of the disease to the offspring through affected
parents and to reduce the prevalence of the disease in the whole breed.
The fact that many inherited canine cataracts develop in the adolescence or the
adulthood, often after the age of first breeding, decreases the effectiveness of this
approach. In case of a recessive mode of inheritance, it is even more difficult to
remove the defective allele from the population, because it can spread among the
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
54
population unnoticed over several generations. It can only be noticed if both parents
pass it to their offspring. Inbreeding increases the risk for infesting with CAT.
As the German Pinscher is a breed predisposed to primary non-congenital cataracts
it is assumed that these cataracts are hereditary. A study in Finland found that a
recessive mode of inheritance seems to be more likely than a dominant mode27. A
DNA test, showing if the dog is homozygous for a CAT-causing mutation or a
heterozygous carrier or free from CAT-causing mutations would be very helpful.
Combined with an adequate breeding program, the prevalence of CAT could be
faster and more effectively decreased in this breed. In addition, the risk of a
selection-caused bottle-neck-phenomenon could be minimized.
The aim of this study was to characterize the prevalence and formation of CAT in the
German Pinscher population in Germany and to analyze the mode of inheritance in
this dog breed.
3.3 Material and Methods
The Pinscher-Schnauzer-Klub 1895 e.V. (PSK) and the German panel of the
European Eye Scheme for diagnosis of inherited eye diseases in animals, the
Dortmunder Kreis (DOK), provided the data for this analysis. The study is based on
the veterinary records for presumed inherited eye diseases of 261 German
Pinschers; the data were collected between January 1995 and October 2009. All
dogs were born in 1993 - 2008. The mean number of German Pinscher puppies per
year (1993 – 2008) was 382; consequently only 7% of all dogs born in this specified
period were ophthalmologically examined until October 2009. The examinations were
performed by specialized and DOK-certified ophthalmologists using slit-lamp
biomicroscopy and indirect ophthalmoscopy. The DOK-members were approved for
examination for presumed inherited eye diseases (PIED) after successful completion
of a two-year training program and examination according to the rules of the
European College of Veterinary Ophthalmologists (ECVO). For all dogs the results
were recorded on official forms based on the standardized eye examination
corresponding to the ECVO. The ECVO considers all bilateral or unilateral cataracts
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
55
and especially cortically cataracts to be hereditary. Exceptions include cases of
obvious association with trauma, inflammation, metabolic disease or nutritional
deficiencies, and minor, clearly circumscript cataracts located in the suture lines or
the nucleus.
For this study all German Pinschers officially diagnosed as affected by or suspicious
for CAT were classified as affected by CAT. All other German Pinschers were
classified as not affected by CAT. The data consisted of a total of 443 examination
reports of 261 dogs. These examinations were performed by 61 ophthalmologists,
each of them carried out from one to 62 examinations (average: 7.26 ± 9.35
examinations). The information from the recording forms was linked with the pedigree
data provided by the PSK.
The pedigree was used for simple segregation analysis to test whether the data were
compatible with the respective simple Mendelian model, a recessive mode of
inheritance. The Singles method is a very straightforward method of simple
segregation analysis28. The test involves a statistical comparison of the estimated
segregation frequency p (probability that an offspring is affected by the respective
disease) with the hypothesized value (p0) arising from the particular model of
inheritance being tested. If a recessive mode of inheritance is assumed, and both
parents in each of a set of full sib families are unaffected, the null hypothesis is that
the true value of p0 = 0.25. The most straightforward use of the Singles method can
be made when the investigator is certain that all families with affected offspring are
included in the data. Then the segregation frequency can be estimated as p = (A -
A1) / (T - A1) and its estimated variance is given by Est. Var. (p) = (T - A) / (T - A1)3 [A
- A1 + 2A2 (T - A) / (T - A1)] where A is the total number of affected offspring in the
available data, T is the total number of all examined offspring in the available data, A1
is the total number of families with just one affected offspring, and A2 is the total
number of families with two affected offspring. Then the null hypothesis is tested
using: Z2 = (p - p0)2 / Est. Var. (p). If the calculated value Z2 is not significant for p0 =
0.25 at α = 0.05, the data are consistent with a simple recessive mode of inheritance.
The mean coefficient of relationship was calculated using OPTI-MATE29 for the group
of German Pinschers with CAT-affected offspring (n = 59) and a contemporary group
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
56
of dogs born in 2008 (n = 555). The mean coefficient of inbreeding was compared
among CAT-affected dogs and the same contemporary group of dogs. Pedigree
information over eight generations was considered, the completeness of pedigrees
was larger then 95% in all eight generations. P values were calculated using t tests.
3.4 Results
Prevalence of CAT
The diagnosis CAT was made in 50 examinations (11.29% of all examinations) of 40
different dogs (15.33% of all dogs). Seven of these 40 dogs were examined one or
two more times after the first diagnosis of CAT was made. Out of the affected
German Pinschers, 67% were diagnosed as affected by CAT in the course of their
first registered ophthalmologic examination.
CAT appeared unilaterally in 9 (22.5%) and bilaterally in 25 (62.50%) cases. CAT
was specified as anterior cortical CAT (55%), posterior polar CAT (15%), nuclear
CAT (2.5%), anterior cortical and posterior polar CAT (10%) or anterior cortical and
posterior polar and nuclear CAT (7.5%). Specification of CAT was missing for four
dogs.
The mean age of onset of CAT was 3.8 ± 1.6 years; the median age of onset was 3.9
years. The youngest reported CAT case was 0.34 years old; the oldest reported CAT
case was 7.44 years old when the first diagnosis of CAT was made. Figure 1 shows
the cumulative distribution of age at diagnosis of CAT in all examined German
Pinschers. Almost 60% of the CAT-affected dogs were diagnosed as affected by
primary CAT up to an age of 4 years. Only 5% were diagnosed as affected by
primary CAT over an age of 6.8 years. CAT was diagnosed in 16/116 (13.68%) of all
males and 24/145 (16.67%) of all females.
The majority of the dogs were examined only once (159/261; 60.92%), 54/261
(20.69%) were examined twice, 28/261 (10.73%) were examined three times and
20/261 (7.66%) were examined four times or more. Age at examination varied
between 0.34 and 13.77 years. Most of the dogs were presented at an age of two to
five years. The first examination for PIED took place on average at 3.0 ± 2.0 years of
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
57
age; when dogs were examined more than once the last registered examination took
place at 4.0 ± 2.3 years of age. About 25% of all dogs were examined at least once
when they were over five years of age. The number of examined males (44.4%) and
females (55.56%) was not significantly different. Dogs originated from 83 kennels.
Pedigree analysis
Investigation of the pedigrees showed close relationships among the dogs affected
by CAT (Fig. 2). There were 58 ophthalmologically examined dogs included in this
pedigree with 20 unaffected and 38 CAT-affected dogs. Of all affected dogs, only two
individuals were not included in this pedigree because of a more distant relationship.
The phenotype of the other 67 dogs was unknown, or they were examined < 4 years
of age. Both males and females had been diagnosed with CAT, which gives reason
to rule out sex linkage. CAT was not found in every generation, which suggests
recessive inheritance for the disease.
The mean coefficient of inbreeding of CAT affected German Pinschers was 7.65 %
which is significantly higher than the mean coefficient of inbreeding of the
contemporary group (3.56 %, Table 1). The mean coefficient of relationship among
parents with at least one CAT affected progeny was with a value of 12.97 %
significantly higher than the mean coefficient of relationship of the contemporary
group (7.44 %). The result of the simple segregation analysis using the Singles
method revealed a Z2 of 0.282 which was not significant at α = 0.05. Accordingly, the
observed distribution of CAT was consistent with a simple recessive mode of
inheritance and a segregation frequency of p = 0.303.
Persistent hyperplastic tunica vasculosa lentis (PHTVL), the ocular disease which
was found most frequently in the Finnish study27 (9% of all examined dogs), was
found in only 4.2% of the German Pinschers examined in the present study. Two
dogs were affected by both diseases. Two other dogs affected by PHTVL were full
sibs of CAT-affected dogs (Fig. 2); the 7 remaining PHTVL-affected individuals were
not included in this pedigree because of a distant relationship to the CAT-affected
dogs and among each other.
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
58
3.5 Discussion
Compared to a large number of dogs of other breeds in Germany, the prevalence of
CAT in German Pinschers in Germany was significantly higher, thus suggesting that
CAT is hereditary in this dog breed. In the present study, the determined prevalence
was higher and the age of onset for CAT lower than in a Finnish study about
cataracts in German Pinschers in Finland27. In the German population the age of
onset was nearly halved compared to the Finnish study. Moreover, it may be
suspected that the true age of onset for CAT in German Pinschers may even be
lower than in the present study because most of the dogs had signs of CAT in their
first ophthalmological examination. Furthermore, in the German population an
anterior cortical CAT was most prevalent whereas in the Finnish study posterior
subcapsular and anterior CATs were found. So we may conclude that there are two
different types of cataracts segregating: German Pinschers with different age of
onset and different prevalences in different populations.
The prevalence of PHTVL in the present study was halved compared to the
prevalence of PHTVL in the Finnish study27. Coherence of all PHTVL-affected
individuals could not be shown in one pedigree because of a more distant
relationship among the individuals.
Inbreeding coefficient was found to have a significant effect on the prevalence of
CAT. In single colored English Cocker Spaniels CAT-affected dogs also had higher
inbreeding coefficients due to matings among carriers and affected dogs30.
Particularly in the presence of a recessive inheritance of CAT, inbreeding among
unaffected carrier animals may lead to an accumulation of alleles identical by
descent. Inbred individuals tend to be homozygous at more loci than no inbred
individuals and in addition, are more likely to be homozygous at the CAT locus.
Comparison of coefficients of relationship revealed a significantly higher mean
relationship coefficient among affected dogs than among a contemporary group of
unaffected dogs. The general high inbreeding level in the whole population of
German Pinschers studied here may be assumed to have significantly contributed to
the increase of CAT-affected dogs because a high number of carriers and in some
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
59
instances CAT-affected dogs were mated. Furthermore, inbreeding decreases
genetic diversity and due to an accumulation of detrimental or defective alleles in an
increasing number of breeding animals, inherited diseases become evident in even
more animals in fewer generations.
The problem of most genetic studies for evaluation of the mode of inheritance for
CAT in dogs is the limited numbers of mating and affected offspring19. Most CATs do
not become manifest until adulthood and may therefore necessitate several years of
observations. However, in many cases assumptions on the mode of inheritance were
based on visual inspection of available pedigrees. In this study, distribution of
information on ophthalmologic examinations allowed simple segregation analyses.
However, the results of this simple segregation analysis do not preclude other more
complex modes of inheritance.
Most congenital cataracts in dog breeds are believed to be inherited as autosomal
monogenic recessive traits, for example in the Welsh Springer Spaniel18, Boston
Terrier20, Miniature Schnauzer20, single colored English Cocker Spaniel30,
Staffordshire bull terrier31, Boston terrier31 and French bulldog31, on the other hand,
an autosomal monogenic incomplete dominant inheritance has been reported in the
Norwegian Buhund17. Furthermore, a dominant mode of inheritance is suspected in
the German Shepherd Dog20 and the Australian Shepherd31 and a polygenic
inheritance of congenital cataracts has been proposed in the Cocker Spaniel23.
The late onset of cataract signs in some of the affected dogs is a big problem for
breeders, as many dogs were used for breeding before CAT was diagnosed.
Regular ophthalmologic examinations, carried out by specialized ophthalmologists,
should be made compulsatory for the assignation of the breeding allowance to
reduce the risk of transmission and the prevalence of the disease in the whole breed
and should be repeated annually as long as the dog is used for breeding. No dogs
with CAT should be used for breeding any more, and breeders should preferably
breed with animals that are old enough to enable positive CAT findings.
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
60
3.6 Acknowledgements
This study was supported by the Pinscher-Schnauzer-Klub e.V. (PSK) and the
Association for Diagnosis of Inherited Eye Diseases in Animals (DOK).
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
61
3.7 References
1. Heinrich CL, Lakhani KH, Featherstone HJ, Barnett KC. Cataract in the UK
Leonberger population. Veterinary Ophthalmology 2006; 9: 350-356.
2. Heitmann M, Hamann H, Brahm R, Grußendorf H, Rosenhagen CU, Distl O.
Analysis of prevalences of presumed inherited eye diseases in Entlebucher
Mountain Dogs. Veterinary Ophthalmology 2005; 8: 145-151.
3. Davidson MG, Nelms SG. Diseases of the lens and cataract formation. In:
Veterinary Ophthalmology 3rd edition (ed. Gelatt KN), Williams & Wilkins:
Philadelphia, 1999; 797-826.
4. Spiess BM. Vererbte Augenkrankheiten beim Entlebucher Sennenhund.
Schweizer Archiv für Augenheilkunde 1994: 136: 105-110.
5. Gelatt KN, Wallace MR, Andrew SE, MacKay EO, Samuelson DA. Cataracts in
the Bichon Frise. Veterinary Ophthalmology 2003; 6: 3-9.
6. Gelatt KN, MacKay EO. Prevalence of primary breed-related cataracts in the dog
in North America. Veterinary Ophthalmology 2005; 8: 101-111.
7. Ketteritzsch K, Hamann H, Brahm R, Grußendorf H, Rosenhagen CU, Distl O.
Genetic analysis of presumed inherited eye diseases in Tibetan Terriers. The
Veterinary Journal 2004; 168: 151-159.
8. Collins BK, Collier LL, Johnson GS, Shibuya H, Moore CP, da Silva Curiel JMA.
Familial cataracts and concurrent ocular anomalies in Chow Chows. Journal of
the American Veterinary Medical Association 1992; 200: 1485-1491.
9. Curtis R, Barnett KC. A survey of cataracts in Golden and Labrador Retrievers.
Journal of Small Animal Practice 1989; 36: 277-286.
10. Barnett KC. Hereditary cataract in the dog. Journal of Small Animal Practice
1978; 19: 109-120.
11. Rubin LF. Cataract in Golden Retrievers. Journal of the American Veterinary
Medical Association 1974; 165: 457-458.
12. Gelatt KN. Cataracts in the Golden Retriever dog. Journal of Veterinary Medical
Education 1972; 67: 1113-1115.
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
62
13. Barnett KC. Hereditary cataract in the German Shepherd Dog. Journal of Small
Animal Practice 1986; 27: 387-395.
14. Barnett KC, Startup FG. Hereditary cataract in the Standard Poodle. The
Veterinary Record 1985 a; 117: 15-16.
15. Barnett KC. Hereditary cataract in the Miniature Schnauzer. Journal of Small
Animal Practice 1985 b; 26: 635-644.
16. Gelatt KN, Samuelson DA, Bauer JE, Das ND, Wolf ED, Barrie KP, Andresen TL.
Inheritance of congenital cataracts and microphthalmia in the Miniature
Schnauzer. American Journal of Veterinary Research 1983; 44: 1130-1132.
17. Narfström K. Cataract in the West Highland White Terrier. Journal of Small
Animal Practice 1981; 22: 467-471.
18. Barnett KC. Hereditary cataract in the Welsh Springer Spaniel. Journal of Small
Animal Practice 1980; 21: 621-625.
19. Gelatt KN, Whitley RD, Lavach JD, Barrie KP, Williams LW. Cataracts in
Chesapeake Bay Retrievers. Journal of the American Veterinary Medical
Association 1979; 175: 1176-1178.
20. Barnett KC. Hereditary cataract in the dog. Journal of Small Animal Practice
1978; 19: 109-120.
21. Yakely WL, Hegreberg GA, Padgett GA. Familial cataracts in the American
Cocker Spaniel. Journal of the American Animal Hospital Association 1971; 39:
127-135.
22. Yakely WL. A study of heritability of cataracts in the American Cocker Spaniel.
Journal of the American Animal Hospital Association 1978; 39: 814-817.
23. Olesen HP, Jensen OA, Norn MS. Congenital hereditary cataract in English
Cocker Spaniel. Journal of Small Animal Practice 1974; 15: 741-750.
24. Roberts SR, Helper L. Cataracts in the Afghan Hounds. Journal of the American
Veterinary Medical Association 1972; 160: 427-432.
25. Koch SA. Cataracts in interrelated Old English Sheepdogs. Journal of the
American Veterinary Medical Association 1972; 160: 299-301.
26. Heywood R. Juvenile cataracts in the Beagle dog. Journal of Small Animal
Practice 1971; 12: 171-177.
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
63
27. Leppänen M, Martenson J, Mäki K. Results of ophthalmologic screening
examinations of German Pinscher in Finland – a retrospective study. Veterinary
Ophthalmology 2001; 4: 165-169.
28. Davie AM. The singles method for segregation analysis under incomplete
ascertainment. Annals of Human Genetics 1979; 42: 507-512.
29. Wrede J, Schmidt T. OPTI-MATE Version 3.81. A management programme
useful to minimize inbreeding in endangered populations. Programme Manual
2003. Institute for Animal Breeding and Genetics, University of Veterinary
Medicine Hannover, Germany.
30. Engelhardt A, Stock KF, Hamann H, Brahm R, Grußendorf H, Rosenhagen CU,
Distl O. A retrospective study on the prevalence of primary cataracts in two
pedigrees from the German population of English Cocker Spaniels. Veterinary
Ophthalmology 2008; 11: 215-221.
31. Mellersh CS, McLaughlin B, Ahonen S, Pettitt L, Lohi H, Barnett KC. Mutation in
HSF4 is associated with hereditary cataract in the Australian Shepherd.
Veterinary Ophthalmology 2009; 12: 372-378.
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
64
3.8 Appendix
Table 1: Comparison of coefficients of inbreeding among CAT affected and a
contemporary group of German Pinscher as well as and relationship coefficients
among parents with CAT affected progeny and a contemporary group of German
Pinschers
Parameter Contemporary
group CAT-affected or German Pinschers
with CAT-affected offspring p-value
Mean coefficient of
relationship (%) 7.44 ± 7.45 12.97 ± 9.46 <0.001
Mean coefficient of
inbreeding (%) 3.56 ± 2.98 7.65 ± 5.68 <0.001
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
65
Figure 1: Cumulative distribution of age at diagnosis in German Pinschers affected
by primary non-congenital cataracts (n = 40)
Prevalence and formation of primary cataracts in the German Pinscher Population in Germany
66
Figure 2: Pedigree showing German Pinschers affected by primary non-congenital
cataracts
CHAPTER 5
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
Julia Menzel, Ute Philipp, Ottmar Distl
Institute for Animal Breeding and Genetics, University of Veterinary Medicine
Hannover, Foundation, Bünteweg 17p, 30559 Hannover, Germany
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
68
4 Scanning 20 candidate genes for association with primary cataracts in German Pinschers
4.1 Abstract
Primary non-congenital cataracts (CAT) are breed related eye diseases and common
in many dog breeds. In this study, we genotyped 36 cataract candidate genes
flanking microsatellite markers for 20 cataract candidate genes in five German
Pinscher families with a total of 45 individuals and tested them for linkage and
association. For delimitation of a linked chromosome region on canine chromosome
28 (CFA28), further eight microsatellites were genotyped on this chromosome was
performed. We sequenced all exons with their flanking intronic regions of PITX3.
Genome-wide significant linkage was found for PITX3-associated Markers.
4.2 Introduction
Primary hereditary cataracts are common in purebred dogs, affecting over 120 dog
breeds worldwide. Cataracts are a frequent cause of visual impairment and blindness
in dogs (Davidson and Nelms 1999, Gelatt and MacKay 2005). Inheritance of non-
congenital cataracts has been demonstrated in several dog breeds, e.g. the Golden
and Labrador Retrievers (Rubin and Flowers 1974, Curtis and Barnett 1989),
German Shepherd (Barnett 1986), West Highland White Terrier (Narfström 1981),
American Cocker Spaniel (Yakely 1978), Tibetan Terrier (Ketteritzsch et al. 2004),
Afghan Hound (Roberts and Helper 1972), Standard Poodle (Rubin and Flowers
1972, Barnett and Startup, 1985), and Entlebucher Mountain Dog (Heitmann et al.
2005). The German Pinscher was shown as a breed predisposed to primary non-
congenital cataracts (CAT) (Lepännen et al. 2001). Pedigree analysis indicated a
monogenic autosomal recessive mode of inheritance for CAT (Menzel and Distl,
2010). The prevalence of CAT in the German Pinscher population in Germany has
been estimated at 15.33% (Menzel and Distl, 2010). The majority of the affected
German Pinschers develop bilateral cataracts, which are mostly located in the
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
69
anterior cortical part of the lens. First signs of CAT in the German Pinscher
population in Germany were registered at a mean age of 3.8 ± 1.6 years.
Because of the relatively late onset of CAT, it is difficult to exclude either CAT
susceptible affected animals early in life from breeding or to ascertain unaffected
carriers. A DNA test, which shows whether the dog is homozygous for a CAT-causing
mutation or a heterozygous carrier or free from CAT-causing mutations, would be
very helpful. Combined with an adequate breeding program, the prevalence of CAT
could be effectively decreased in this breed.
To date, more than 20 genes that have to be considered as possible candidate
genes for primary cataracts in dogs because these genes were found to be
associated with hereditary cataracts in humans or mice (Reddy et al. 2004, Graw
2004, Hunter et al. 2006, Mellersh et al. 2006, Müller and Distl 2009). These genes
encode structural or membrane transport proteins of the lens, transcription factors
which are involved in eye and lens development, or enzymes which are necessary
for lens metabolism (Graw 2004). Here, we investigated the 20 genes reported by
Mellersh et al. (2006) and Müller and Distl (2009).
The candidate gene on CFA28, the paired-like homeodomain 3 (PITX3) gene,
encodes a member of the RIEG/PITX homeobox family, which is in the bicoid class of
homeodomain proteins. Members of this family act as transcription factors. This
protein is involved in lens formation during eye development. Mutations of this gene
have been associated with anterior segment mesenchymal dysgenesis and
congenital cataracts in humans and mice (Semina et al. 1997, Semina et al. 1998,
Rieger et al. 2001, Medina-Martinez et al. 2009). The PITX3 gene is conserved in
chimpanzee, dog, cow, mouse, and rat. These observations made PITX3 a candidate
for CAT in dogs.
4.3 Material and Methods
Animals, phenotypic data and DNA specimens
Ophthalmological data for the German Pinschers were provided by the Dortmunder
Kreis (DOK), the German panel of the European Eye Scheme for diagnosis of
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
70
inherited eye diseases in animals. The ophthalmological examinations of the
investigated dogs were carried out by veterinary specialists of the DOK and in
accordance with the rules of the European College of Veterinary Ophthalmologists
(ECVO). Only primary cataracts, and not secondary cataracts caused by diabetes or
trauma, were recorded.
The Pinscher-Schnauzer-Klub e.V. (PSK) supplied pedigree data and we identified
pedigrees with multiple CAT-affected dogs. For the present analysis, we chose 45
dogs from five different German Pinscher families (Figure 1). Altogether this study
included 15 CAT-affected German Pinschers. In most of the affected dogs included
in this analysis, the opafication of the lens was located in the anterior cortex. Thirteen
(86.67%) of these 15 dogs had an anterior cortical cataract. Both eyes were affected
in 11 animals (73.33%), while alterations were found only in the lens of the left or the
right eye in the other four. Most of the dogs (about 70%) were examined two or three
times. At least one unaffected dog was investigated from each family. Table 1 shows
the distribution of localization and status of CAT for the 5 German Pinscher families
included in our analyses. The 14 unaffected dogs were over 4.6 years old at the last
ophthalmological examination. Unaffected dogs with the last ophthalmological
examination at an age <4.5 years were classified as dogs with unknown phenotype
(Table 1).
We also tested three unaffected dogs from other breeds as control animals. Two
milliliters EDTA blood (BIOTA) was obtained from each dog, and DNA was extracted
using QIAamp 96 DNA Blood kit (Qiagen, Hilden, Germany).
Genotyping of microsatellites
The microsatellites were in a distance to the particular candidate genes of less than
one megabase (Mb). For the investigation of the 20 candidate genes we used
microsatellites and PCR conditions according to Mellersh et al. (2006) and Müller and
Distl (2009). We employed the microsatellites LIM2_1_107.53, LIM2_1_108.39,
FTL_1_109.84, FTL_1_109.88, CRYAB_5_24.38, MAF_5_74.57, MAF_5_75.09,
HSF4_5_85.24, HSF4_5_85.60, CRYBA1_9_35.59, CRYBA1_9_36.14,
MIP_10_3.58, MIP_10_3.77, FOXE3_15_16.23, FOXE3_15_16.33, GJA8_17_61.25,
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
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PAX6_18_46.18, PAX6_18_46.45, TRNT1_20_16.80, TRNT1_20_17.24,
BFSP2_23_33.31, BFSP2_23_33.67, GJA3_25_20.87, GJA3_25_21.70,
CRYBB2_26_23.50, CRYBB2_26_23.59, PITX3_28_16.97, EYA1_29_23.05,
SORD_30_14.41, SORD_30_14.58, CRYAA_31_39.03, CRYAA_31_39.65,
GCNT2_35_13.04, GCNT2_35_13.64, CRYGA_37_19.28 and CRYGA_37_19.59.
For fine mapping of the linked region on canine chromosome 28 (CFA28), we chose
three additional microsatellites from the Minimal Screening Set 2 (Guyon et al. 2003;
Clark et al. 2004) (C28176, FH2585, REN51i12) and five additional markers from the
dog genome assembly 2.1 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi):
CF28_11.36, CF28_16.26, CF28_17.26, CF28_17.89 and CF28_19.99.
The PCR primers and conditions are shown in Table 2. The PCR for genotyping of
the microsatellites started at 94°C for 4 min, followed by 38 cycles at 94°C for 30 sec,
optimum annealing temperature for 1 min, 72°C for 30 sec, and at 4°C for 10 min. All
PCR reactions were performed in 11.5-µl reactions using 6 pmol of each primer, 0.2
µl dNTPs (100 µM) and 0.1 µl Taq-DNA-Polymerase (5 U/µl) (Q-Biogen, Heidelberg,
Germany) in the reaction buffer supplied by the manufacturer for 2 µl template DNA.
The forward primers were labelled fluorescently with IRD700 or IRD800. For the
analysis of the marker genotypes, PCR products were size-fractionated by gel
electrophoresis on an automated sequencer (LI-COR, Lincoln, NE, USA) using 4%
polyacrylamide denaturing gels (Rotiphorese Gel40, Carl Roth, Karlsruhe). Allele
sizes were detected using an IRD700- and IRD800-labeled DNA ladder; the
genotypes were assigned by visual examination.
Non-parametric linkage analysis
A non-parametric multipoint linkage analysis was employed for the German Pinscher
families using the MERLIN 1.1.2 (Abecasis et al. 2002). This analysis is based on
allele sharing among affected individuals by identical-by-descent methods (Kong and
Cox 1997). Haplotypes were estimated using MERLIN 1.1.2 with the option “best”. A
case-control analysis based on χ2-tests for genotypes, alleles and trend of the most
prevalent allele was also performed for the German Pinschers. The CASECONTROL
and ALLELE procedures of SAS/Genetics were used for association tests, tests for
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
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Hardy-Weinberg equilibrium of genotype frequencies and the estimation of allele
frequencies (SAS Institute, 2010).
Structural and mutation analysis of PITX3
We searched the dog-expressed sequence tag (EST) archive
(http://www.ncbi.nlm.nih.gov/genome/seq/CfaBlast.html) for ESTs by cross-species
BLAST searches with the corresponding human reference mRNA sequences for
PITX3 (NM_005029.3). We found a canine EST (DN379690.1) isolated from dog
synovial tissue with 90% identity to the human PITX3 mRNA sequence. A significant
match to this canine EST was identified on canine chromosome 28 by means of
BLASTN searches of the canine EST against the dog genome assembly (Dog
genome assembly 2.1). The genomic structure of the canine PITX3 gene was
determined with the Spidey mRNA-to-genomic alignment program
(http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey/index.html).
We sequenced all exons and the flanking intronic regions of the canine PITX3 gene
for 6 affected and 4 unaffected German Pinschers out of the five families. PCR
primers were designed using the Primer3 program (http://frodo.wi.mit.edu/cgi-
bin/primer3_www.cgi) based on the canine EST (DN379690.1) and the genomic
sequence for canine PITX3 (LOC 486856). The PCR primers are listed in Table 3. All
PCRs were performed in 50-µl reactions using 20 pmol of each primer, 40 µM
dNTPs, 0.5 U KappG-Robust-DNA-Polymerase (PeqLab, Erlangen, Germany) in the
reaction buffer supplied by the manufacturer, 5x PCR Enhancer 1 (PeqLab,
Erlangen, Germany), and 5% DMSO for 3 µl template DNA. The PCR conditions
were: 95°C for 5 min followed by 38 cycles of 95 °C for 30 s, optimum annealing
temperature for 30 s, 72°C for optimum elongation time, and 4°C for 10 min. All PCR
products were cleaned using the Nucleo-Fast PCR purification kit (Macherey-Nagel)
and directly sequenced with the DYEnamic ET Terminator kit (GE healthcare,
München, Germany) and a MegaBACE 1000 capillary sequencer (GE Healthcare).
Sequence data were analysed with Sequencher version 4.7 (GeneCodes, Ann
Arboer, MI, USA).
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4.4 Results and Discussion
Non-parametric linkage analysis
Table 4 shows the results of the non-parametric linkage analysis for all candidate
gene flanking microsatellites. The highest and the only significant LOD score of 0.64
was obtained for the marker PITX3_28_16.97 which is located next to the candidate
gene PITX3 on CFA28. We genotyped eight additional microsatellite markers on
CFA28 to verify the linkage of these markers and to delimit the linked region on
CFA28. After that, the highest LOD scores were obtained for the markers
CF28_16.26, PITX3_28_16.97, CF28_17.26 and CF28_17.89, which are located in a
region of 0.5 Mb proximal and 1.1 Mb distal of the PITX3 gene (Table 5). The
maximum achievable Zmean was 7.05 and the corresponding value for the LOD
score was 2.50 indicating that the power of the analysis was high enough to detect
genome-wide significant linkage. The error probabilities for linked markers ranged
from 0.015 to 0.02. The polymorphism information content of the individual markers
was between 60.92 and 77.54%.
Structural and mutation analysis of PITX3
The canine EST for PITX3 (DN379690.1), which was found by cross-species BLAST
searches with the corresponding human reference mRNA sequences, mapped to the
same position as the annotated canine gene for PITX3 (LOC486856) and revealed
an open reading frame of 1029 bp predicting a protein of 302 amino acids (Fig. 2).
We performed a mutation analysis for the PITX3 gene due to the significant linkage
of the flanking markers. The canine PITX3 gene consists of four exons interrupted by
one long (intron 1) and two short introns. The sequencing of exon 2 and 3 failed
partly despite repeated attempts with different PCR conditions, the cause could be
the abundance GC. The search for sequence variations within exon 1 and 4 and
parts of exon 2 and 3 of this gene revealed a total seven single nucleotide
polymorphisms (SNPs) as shown in table 6. Of these seven SNPs, five were located
in the exon 3 sequence of PITX3 (g322T>C, g.324G>C, g.334T>C, g.336G>C,
g.352G>C), while the others were located in intron 2 next to 5’ of exon 3 (g.309T>C,
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
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g.311G>C). Two of the exonic SNPs (g322T>C, g.334T>C) were T/C transitions and
cause an amino acid change from serin (S) to tryptophan (W) compared to the dog
reference sequence (boxer). In the protein sequences of human and mice, the same
protein sequence as in the German Pinscher were found (Fig. 6). The other exonic
SNPs (g.324G>C, g.336G>C, g.352G>C) were transitions without cause of amino
acid change. We found no polymorphisms within the German Pinscher breed.
Sequencing of the complete exons 2 and 3 for all animals should be performed in
order to detect polymorphisms in these regions which could be possibly associated
with the CAT phenotype in the German Pinscher.
Another approach is the analysis of cDNA of the PITX3 gene. Lens tissue of German
Pinscher affected by CAT should be collected in connection with cataract surgery, for
example with the phacoemulsification method with ultrasound. After removal from the
eye, the lens tissue could be conserved using RNA-later solution.
To date, there are no other cataract candidate genes known in the linked region, and
we could not find any possible functional cataract candidate genes by searching this
region in the current dog genome assembly 2.1 (http://www.ncbi.
nlm.nih.gov/mapview/mapsearch.cgi?taxid=9615).
4.6 Acknowledgements
The authors express their gratitude to the Pinscher-Schnauzer-Klub 1895 e.V. for
providing the pedigree data and the blood samples. The authors would like to thank
the veterinary ophthalmologists of the Dortmunder Kreis (DOK) for providing the
ophthalmological data.
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4.7 References
Abecasis GR, Cherny SS, Cookson WO, Cardon LR. Merlin-rapid analysis of dense
genetic maps using sparse gene flow trees. Nature Genetics 2002; 30: 97-101.
Barnett KC. Hereditary cataract in the German Shepherd Dog. Journal of Small
Animal Practice 1986; 27: 387-395.
Barnett KC, Startup FG. Hereditary cataract in the Standard Poodle. The Veterinary
Record 1985 a; 117: 15-16.
Clark LA, Tsai KL, Steiner JM, Williams DA, Guerra T, Ostrander EA, Galibert F,
Murphy KE. Chromosome-specific microsatellite multiplex sets for linkage studies
in the domestic dog. Genomics 2004; 84: 550–554.
Curtis R, Barnett KC. A survey of cataracts in Golden and Labrador Retrievers.
Journal of Small Animal Practice 1989; 36: 277-286.
Davidson MG, Nelms SG. Diseases of the lens and cataract formation. In: Veterinary
Ophthalmology 3rd edition (ed. Gelatt KN), Williams & Wilkins: Philadelphia, 1999;
797-826.
Gelatt KN, MacKay EO. Prevalence of primary breed-related cataracts in the dog in
North America. Veterinary Ophthalmology 2005; 8: 101-111.
Graw J. Congenital hereditary cataracts. International Journal of Developmental
Biolology 2004; 48: 1031-1044.
Guyon R, Lorentzen TD, Hitte C, Kim L, Cadieu E, Parker HG, Quignon P, Lowe JK,
Renier C, Gelfenbeyn B, et al. A 1-Mb resolution radiation hybrid map of the
canine genome. Proceedings of the National Academy of Sciences of the United
States of America 2003; 100: 5296–5301.
Heitmann M, Hamann H, Brahm R, Grußendorf H, Rosenhagen CU, Distl O. Analysis
of prevalences of presumed inherited eye diseases in Entlebucher Mountain
Dogs. Veterinary Ophthalmology 2005; 8: 145-151.
Hunter LS, Sidjani DJ, Johnson JL, Zangerl B, Galibert F, Andre C, Kirkness E,
Talamas E, Acland GM, Aguirre GD. Radiation hybrid mapping of cataract genes
in the dog. Molcular Vision 2006; 12: 588-596.
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
76
Ketteritzsch K, Hamann H, Brahm R, Grußendorf H, Rosenhagen CU, Distl O.
Genetic analysis of presumed inherited eye diseases in Tibetan Terriers. The
Veterinary Journal 2004; 168: 151-159.
Kong A, Cox NJ. Allele-sharing models: LOD scores and accurate linkage tests. The
American Journal of Human Genetics 1997; 61: 1179-1188.
Leppänen M, Martenson J, Mäki K. Results of ophthalmologic screening
examinations of German Pinscher in Finland – a retrospective study. Veterinary
Ophthalmology 2001; 4: 165-169.
Medina-Martinez O, Shah R, Jamrich M. Pitx3 controls multiple aspects of lens
development. Developmental Dynamics 2009; 238: 2193-2201.
Mellersh CS, Pettitt L, Forman OP, Vaudin M, Barnett KC. Identification of mutations
in HSF4 in dogs of three different breeds with hereditary cataracts. Veterinary
Ophthalmology 2006; 9: 369-378.
Narfström K. Cataract in the West Highland White Terrier. Journal of Small Animal
Practice 1981; 22: 467-471.
Reddy MA, Francis PJ, Berry V, Bhattacharya SS, Moore AT. Molecular genetic
basis of inherited cataract and associated phenotypes. Survey of Ophthalmology
2004; 49: 300-315.
Rieger DK, Reichenberger E, McLean W, Sidow A, Olsen BR. A double-deletion
mutation in the Pitx3 gene causes arrested lens development in aphakia mice.
Genomics 2001; 72: 61-72.
Roberts SR, Helper L. Cataracts in the Afghan Hounds. Journal of the American
Veterinary Medical Association 1972; 160: 427-432.
Rubin LF, Flowers RD. Cataract in Golden Retrievers. Journal of the American
Veterinary Medical Association 1974; 165: 457-458.
Rubin LF, Flowers RD. Inherited cataract in a family of Standard Poodles. Journal of
the American Veterinary Medical Association 1972; 161: 207-208.
SAS Institute. SAS/Genetics, Version 9.1.3. Cary, NC, USA, 2005
Semina EV, Reiter RS, Murray JC. Isolation of a new homeobox gene belonging to
the Pitx/Rieg family: expression during lens development and mapping to the
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
77
aphakia region on mouse chromosome 19. Human Molcular Genetics 1997; 6:
2109-2116.
Semina EV, Ferrell RE, Mintz-Hittner HA, Bitoun P, Alward WL, Reiter RS,
Funkhauser C, Daack-Hirsch S, Murray JC. A novel homeobox gene PITX3 is
mutated in families with autosomal-dominant cataracts and ASMD. Nature
Genetics 1998; 19: 167-170.
Yakely WL. A study of heritability of cataracts in the American Cocker Spaniel.
Journal of the American Animal Hospital Association 1978; 39: 814-817.
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
78
4.8 Appendix
Table 1: Distribution of localisation and status of CAT for the five German Pinscher
families (* unaffected dogs with the last ophthalmological examination at an age <4.5
years were classified as dogs with unknown phenotype)
Localisation of the
opafication
Family Number
of
affected
dogs
Number of
unaffected
dogs
Number of
dogs with
unknown
phenotype*
Cortex Capsule or
Nucleus
Both
eyes
affected
Only left
or right
eye
affected
1 4 8 4 4 0 3 1
2 3 1 3 2 1 2 1
3 2 2 0 2 0 2 0
4 2 2 0 2 0 1 1
5 4 1 9 3 1 3 1
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Table 2: PCR primers with their product size range and annealing temperature (Ta)
for the amplification of the additional microsatellites on canine chromosome 28
(CFA28).
Name of
microsatellite
Position on
CFA28
(Mb)
Sequence (5’ – 3’) of primers Ta
(°C)
Size
range
(bp)
TCAGGCTCTCCAGAGACACA C28.176 4.50
TTGCCAATAGCAGACTGTGG
58 186-196
AGTTTGGGCCTGAGAATGTC CF28_11.36 11.36
AGGGAATCATTGGATATGGTG
58 249
AGCTCTTGCTCTCTGTGCTG CF28_16.26 16.26
GCAGTGGAGAAACTTGGTAAGC
59 200
GTTCGCCTGATCCTTACACAA PITX3_28_16.97 16.97
CCCACATGACGCCTAGACTAA
60 300
AATTTATGGCTCCCCTTCAC CF28_17.26 17.26
TTGATCACTGTTCTGCCAAC
58 203
TCATGAAGCTGAGGAGAAAAG CF28_17.89 17.89
CTAACAACTGGGGTCACAGG
58 341
AAACTGTGGAGCACAAAATGG CF28_19.99 19.99
GCCTTTTTATCTTTTCTAATCATGC
59 272
TCGATGTCTGCCTTTCTTGA FH2585 26.01
GCCCCCTCACCTCATATTCT
58 238-334
ACTTTCTTTGAGCGAGGTG REN51i12 37.38
CTGGCCTGTGCTGGGTGAG
55 228-438
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Table 3: PCR primers, their position on canine chromosome 28 (CFA28), their
product size and annealing temperature (Ta) for the amplification of the genomic
sequence of the canine PITX3 gene
Primer Position on
CFA28 (Mb)
Target Sequence (5’ – 3’) of primers Ta
(°C)
Product size
(bp)
CCCAGATGCTGAGGAGTACC
CTGTCCTGGCTCCATGTCTC
60 661
GGAGTACCTCCGATTGGC 641
PITX3_1F
PITX3_1R
PITX3_1IntF
PITX3_1IntR
17.771866 -
17.772526
5’ UTR,
Exon 1,
Intron1
GCTCCATGTCTCTGGCTTC
58
ACAGTACGTGGTGTGCAATG
GCGTGGAGGATAAACACAAG
58
1220
GGAACACGGCTGCAAGG 302
PITX3_2F
PITX3_2R
PITX3_2IntF
PITX3_2IntR
17.762293 -
17.763512
Exon 2,
Intron 2,
Exon 3
CTGCAACTGCTGGCTGGTG
61
CACTGTCCCTCGTCTTCTTC
TACATATTTGCACGCAGGTG
58
1407
TCAACGTGGGGCCTCTG 457
PITX3_3F
PITX3_3R
PITX3_3IntF
PITX3_3IntR
17.761075 -
17.762481
Intron 3,
Exon 4,
3’ UTR
CCAGTCAAGACGACCCCAG
62
Table 4: Non-parametric test statistics Zmean and LOD Score, their error probabilities (PZ, PL), polymorphism information
content (PIC), χ2-tests for allele and genotype distribution of the case-control analysis, degrees of freedom (DF) and their
corresponding error probabilities (P) for the candidate gene flanking microsatellite markers in the German Pinscher
Test for linkage Test for association Gene Marker Position
on CFA
PIC
(%) Zmean PZ LOD
score
PL χ2
geno-
type
DF P
geno-
type
χ2
allele
DF P
allele
LIM2_1_107.53 107.53 64.95 -0.01 0.5 -0.00 0.5 4.60 9 0.86 1.46 3 0.69 LIM2
(CFA1) LIM2_1_108.39 108.39 59.67 -0.37 0.6 -0.05 0.7 5.89 7 0.55 1.14 4 0.88
FTL_1_109.84 109.84 32.49 0.28 0.6 -0.04 0.7 3.24 2 0.19 1.05 1 0.30 FTL
(CFA1) FTL_1_109.88 109.88 52.74 -0.30 0.6 -0.04 0.7 1.90 4 0.75 0.66 2 0.71
CRYAB
(CFA5)
CRYAB_5_24.38 24.38 53.24 0.07 0.5 0.00 0.4 9.22 8 0.32 3.45 4 0.48
MAF_5_74.57 74.57 12.14 0.06 0.5 0.00 0.4 0.70 1 0.40 0.64 1 0.42 MAF
(CFA5) MAF_5_75.09 75.09 37.44 0.06 0.5 0.00 0.4 0.91 2 0.63 0.01 1 0.89
HSF4_5_85.24 85.24 57.42 0.06 0.5 0.00 0.5 4.36 6 0.62 2.48 3 0.47 HSF4
(CFA5) HSF4_5_85.60 85.60 52.49 0.08 0.3 0.01 0.4 3.89 6 0.69 2.73 3 0.43
CRYBA1_9_35.59 35.59 47.82 0.08 0.6 -0.02 0.6 6.35 5 0.27 1.86 3 0.60 CRYBA1
(CFA9) CRYBA1_9_36.14 36.14 53.24 0.08 0.5 0.01 0.4 6.75 5 0.23 2.26 4 0.68
MIP_10_3.58 3.58 73.36 -0.49 0.7 -0.07 0.7 15.38 15 0.42 8.64 7 0.27 MIP
(CFA10) MIP_10_3.77 3.77 16.90 -0.49 0.7 -0.07 0.7 1.76 3 0.62 0.56 2 0.75
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FOXE3_15_16.23 16.23 42.11 -0.29 0.6 -0.04 0.7 1.50 3 0.68 1.23 3 0.53 FOXE3
(CFA15) FOXE3_15_16.33 16.33 54.17 -0.29 0.6 -0.04 0.7 6.31 4 0.17 2.30 2 0.31
GJA8
(CFA17)
GJA8_17_61.25 61.25 53.55 -0.33 0.6 -0.05 0.7 6.50 5 0.25 1.80 5 0.40
PAX6_18_46.18 46.18 47.99 -0.06 0.5 -0.01 0.6 7.49 5 0.18 7.31 3 0.06 PAX6
(CFA18) PAX6_18_46.45 46.45 23.74 -0.05 0.5 -0.00 0.5 7.31 3 0.06 7.20 3 0.06
TRNT1_20_16.80 16.80 59.13 0.33 0.4 0.17 0.2 3.71 5 0.59 1.12 2 0.56 TRNT1
(CFA20) TRNT1_20_17.24 17.24 33.29 0.32 0.4 0.16 0.2 0.59 2 0.74 0.21 1 0.64
BFSP2_23_33.31 33.31 55.28 -0.80 0.8 -0.13 0.8 0.97 5 0.96 0.03 2 0.98 BFSP2
(CFA23) BFSP2_23_33.67 33.67 58.36 -0.80 0.8 -0.13 0.8 0.84 5 0.97 0.09 2 0.95
GJA3_25_20.87 20.87 67.81 -0.52 0.7 -0.08 0.7 14.69 11 0.19 1.14 4 0.88 GJA3
(CFA25) GJA3_25_21.70 21.70 55.38 -0.83 0.8 -0.14 0.8 4.62 6 0.59 0.45 3 0.92
CRYBB2_26_23.50 23.50 48.55 -0.60 0.7 -0.10 0.7 5.15 7 0.64 4.39 4 0.35 CRYBB2
(CFA26) CRYBB2_26_23.59 23.59 48.21 -0.60 0.7 -0.10 0.7 6.68 6 0.35 3.74 3 0.32
PITX3
(CFA28)
PITX3_28_16.97 16.97 65.50 1.18 0.12 0.64 0.04 7.56 9 0.57 2.03 3 0.56
EYA1
(CFA29)
EYA1_29_23.05 23.05 58.81 -0.15 0.6 -0.02 0.6 10.63 7 0.15 5.92 3 0.11
SORD_30_14.41 14.41 57.52 0.29 0.4 0.09 0.3 11.27 9 0.25 6.10 4 0.19 SORD
(CFA30) SORD_30_14.58 14.58 54.75 0.29 0.4 0.09 0.3 5.47 6 0.48 1.23 3 0.74
CRYAA_31_39.03 39.03 51.62 -0.38 0.7 -0.05 0.7 4.59 5 0.46 0.88 3 0.82 CRYAA
(CFA31) CRYAA_31_39.65 39.65 27.07 -0.39 0.7 -0.06 0.7 0.14 2 0.93 0.14 1 0.70
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GCNT2_35_13.04 13.04 52.60 0.06 0.5 0.00 0.5 7.71 7 0.35 1.26 4 0.86 GCNT2
(CFA35) GCNT2_35_13.64 13.64 53.22 0.11 0.4 0.00 0.4 0.45 4 0.97 0.32 3 0.95
CRYGA_37_19.28 19.28 58.66 0.87 0.2 0.38 0.09 2.68 5 0.74 0.43 2 0.80 CRYGA
(CFA37) CRYGA_37_19.59 19.59 39.66 0.86 0.2 0.38 0.09 1.53 5 0.90 0.73 3 0.86
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Table 5: Non-parametric test statistics Zmean and LOD Score, their error probabilities (PZ, PL), polymorphism information
content (PIC), χ2-tests for allele and genotype distribution of the case-control analysis, degrees of freedom (DF) and their
corresponding error probabilities (P) for all microsatellite markers on canine chromosome 28 (CFA28) in the German
Pinscher
Tests for linkage Tests for association Gene Marker Position
on
CFA28
PIC
(%) Zmean PZ LOD
score
PL χ2
geno-
type
DF P
geno-
type
χ2
allele
DF P
allele
C28.176 4.50 63.95 1.08 0.14 0.33 0.11 6.84 8 0.55 0.76 3 0.85
CF28_11.36 11.36 70.70 0.85 0.2 0.21 0.2 15.61 14 0.33 8.99 7 0.25
CF28_16.26 16.26 77.54 2.12 0.02 0.97 0.02 13.50 16 0.63 2.98 5 0.70
PITX3_28_16.97 16.97 65.50 2.34 0.010 1.03 0.015 7.56 9 0.57 2.03 3 0.56
CF28_17.26 17.26 66.02 2.41 0.008 1.03 0.015 5.32 11 0.91 1.63 4 0.80
CF28_17.89 17.89 60.92 2.29 0.011 0.99 0.02 4.66 8 0.79 2.06 3 0.55
CF28_19.99 19.99 63.19 1.47 0.07 0.63 0.04 16.17 12 0.18 11.77 7 0.10
FH2585 26.01 69.60 0.89 0.2 0.40 0.09 9.46 13 0.73 1.14 5 0.95
PITX3
REN51i12 37.38 49.76 0.72 0.2 0.15 0.2 6.52 7 0.47 6.03 5 0.71
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Table 6: Single nucleotide polymorphisms (SNPs) within the German Pinscher for the
canine PITX3 gene
SNP LOC486856 Location Effects on protein sequence g.309T>C Intron 2 no effects g.311G>C Intron 2 no effects g322T>C Exon 3 S>W g.324G>C Exon 3 no effects g.334T>C Exon 3 S>W g.336G>C Exon 3 no effects g.352G>C Exon 3 no effects
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Figure 1: Pedigrees of the five German Pinscher families which were used for the
candidate genes scan for association with primary cataracts.
Haplotypes for the three linked microsatellite markers and their two flanking markers
were given over the symbols for each proband.
First line: CF28_16.26
Second line: PITX3_28_16.97
Third line: CF28_17.26
Fourth line: CF28_17.89
Fifth line: CF28_19.99
Family 1:
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Family 2:
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Family 3:
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
89
Family 4:
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
90
Family 5:
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
91
Figure 2: Alignment of the canine PITX3 protein (302 amino acids) of two different
dog breeds (boxer and German Pinscher (GP)) with the known orthologous protein
sequences. The sequences were derived from GenBank entries with the association
nos. NP_032878 (mouse) and NP_005020 (human). Identical residues to the
German Pinscher are indicated by asterisks. Amino acids 40 (SNP g.322T>C) and 44
(SNP g.334T>C) are indicated by a grey underlay).
GP MEFGLLSEAEARSPALSLSDAGTPHPPLPEHGCKGQEHSDSEKASASLPG 50
boxer ****************************************W***W***** 50
mouse ******G******************************************* 50
human **************************Q*********************** 50
GP GSPEDGSLKKKQRGQRTHFTSQQLQELEAPFQRNRYPDMSTREEIAVWTN 100
boxer *************G************************************ 100
mouse *****************************T******************** 100
human *****************************T******************** 100
GP LTEARVGVWFKNRRAKWRKRERSQQAELCKGGFAAPLGGLVPPYEEVYPG 150
boxer ************************************************** 150
mouse ******R******************************************* 150
human ******R************************S****************** 150
GP YSYGNWPPKALGPPLAAKTFPFAFNSVNVGPLASQPVFSPPSSIAASMVP 200
boxer ************************************************** 200
mouse ***********A************************************** 200
human ***********A************************************** 200
GP SAAAAPGTVPGPGALQGLGGGPPGLAPAAVSSGAVSCPYASAAAAAAAAA 250
boxer ************************************************** 250
mouse ********************A***************************** 250
human ************************************************** 250
Scanning 20 candidate genes for association with primary cataracts in German Pinschers
92
GP SSPYVYRDPCNSSLASLRLKAKQHASFSYPAVPGPPPAANLSPCQYAVER 300
boxer ************************************************** 300
mouse ************************************************** 300
human ********************************H***************** 300
GP PV 302
boxer ** 302
mouse ** 302
human ** 302
CHAPTER 6
General discussion
General discussion
94
6 General discussion
In our population analysis for persistent right aortic arch (PRAA) in the German
Pinscher, which was performed at the beginning of the present thesis, we discovered
a rare combination of vascular anomalies that has only been described in two
isolated cases of other dog breeds before (House et al., 2005). In the German
pinscher, the occurrence of any form of PRAA was not previously known. We can
conclude from our analyses that PRAA is an inherited disease in the German
Pinscher. This study did not analyze the mode inheritance of PRAA in the German
Pinscher but excluded specific forms of inheritance. However, PRAA in other dog
breeds is believed as a complex polygenetic trait (Patterson, 1989). The proof of the
mode of inheritance of PRAA is difficult because of the lack of profound knowledge of
many veterinarians. The consequence is that a lot of puppies with PRAA-suspicious
symptoms get euthanized without correct diagnoses of the PRAA affectation status.
Therefore, a lot of cases of PRAA remain undetected. Further genetic analyses and
better knowledge of diagnosing this type of vascular ring anomaly is necessary to
clarify the mode of inheritance in order to facilitate molecular genetic analyses of
PRAA in the German Pinscher.
Comparative genetic research revealed that conotruncal heart defects in humans
have been associated with deletion of the chromosome 22q11.2 region which is also
known as the Digeorge critical region (DGCR) (Rauch et al., 2004; Lee et al., 2006).
Monosomy 22q11.2 is strongly associated with the presence of anomalies of the
subclavian artery which were found in all three puppies examined. The genes located
within the DGCR on 22q11.2 are almost completely conserved on mouse
chromosome 16. Mice with a heterozygeous deletion of a 1.5-Mb homologous
DiGeorge region show defects similar to those seen in del22q11.2 patients, including
aortic arch anomalies and subclavian artery anomalies. The TBX1 gene, which is
important for cardiomorphogenesis, is located in the DGCR. These observations
made TBX1 a candidate gene for the cardiovascular manifestations of del22q11.2
syndrome.
The candidate gene scan of TBX1 and additional genes on the same chromosome in
General discussion
95
the canine DiGeorge region revealed significant linkage for the flanking marker of the
TBX1 gene with the PRAA phenotype in the German Pinscher. Genotyping of 37
microsatellites on CFA 26 in all dogs delimited the linked region to about 1 Mb. We
sequenced all exons and flanking intronic regions of the candidate gene TBX1 and
parts of other genes in the linked region and found two single nucleotide
polymorphisms (SNPs) in the TBX1 gene to be associated with the PRAA phenotype
in the German Pinscher. These two SNPs were found to be homozygous in all
affected animals and heterozygous in all non-affected animals. This could be an
indication for a deletion in the affected animals in this region.
Because of the small number of probands, further genetic studies with a larger
number of animals are necessary to confirm the role of the TBX1 gene in the
molecular background of PRAA in German Pinschers.
Primary cataracts are a common disease in purebred dogs. More than 120 dog
breeds worldwide are known to be affected by cataracts with presumed or
established inheritance. Cataracts are the leading cause of blindness in purebred
dogs (Helper, 1989; Rubin, 1989; Davidson and Nelms, 1999; Slatter, 2001).
In the present thesis, the German Pinscher was considered for the population genetic
analyses and included in the molecular genetic analyses. Previous studies on
German Pinschers in Finland had revealed the importance of cataracts in these dogs
(Lepännen et al., 2001). In addition, breeders of German pinschers in Germany and
other countries reported problems with primary cataracts and presumed a genetic
background in this dog breed. We found prevalence of primary cataracts of 15.33%
in the German Pinscher population in Germany. The ophthalmological data were
linked with the pedigree data provided by the Pinscher-Schnauzer-Klub 1895 e.V.
and were available for 261 German Pinschers. For primary cataracts (CAT) in the
German Pinscher population in Finland, age of manifestation of nine years has been
reported (Lepännen et al., 2001). Primary cataracts in the German pinscher
population of our study in Germany manifests at a mean age of 3.8 ± 1.6 years. In
the German population the age of onset was nearly halved compared to the Finnish
study (Lepännen et al., 2001). Moreover, it may be suspected that the true age of
General discussion
96
onset for CAT in the German Pinscher may even be lower than in the present study
because most of the dogs had signs of CAT in their first ophthalmological
examination. Furthermore, in the German population CAT was more prevalent and
an anterior cortical CAT was the most prevalent form whereas in the Finnish study
the prevalence was lower and posterior subcapsular and anterior CATs were found.
Therefore we may conclude that there are two different types of cataracts
segregating: German Pinschers with different age of onset and different prevalences
in different populations. The observed distribution of CAT was consistent with a
simple recessive mode of inheritance according to the results of the simple
segregation analysis using the Singles Method. However, the results of this simple
segregation analysis do not preclude other more complex modes of inheritance.
More than 20 genes are known to be involved in the pathogenesis of different types
of CAT in humans and mice (Beby et al., 2003; Graw, 2004; Reddy et al., 2004).
These genes were used for a candidate gene approach. We could exclude most of
them for being responsible for CAT in the German Pinscher. For the flanking
microsatellites of one gene, PITX3, we could detect significant linkage with the CAT
phenotype in the German Pinscher, but the mutation analyses of this gene did not
reveal any possible causal mutations. Sequencing of exon 2 and 3 failed partly;
therefore in these regions undetected mutations are possible. Genotyping of
additional microsatellites on the same chromosome expanded the linked region to
about 1.6 Mb. The highest LOD scores were obtained for markers located from 0.5
Mb proximal to 1.1 Mb distal of PITX3.
To date, there are no other cataract candidate genes known in the linked region, and
we also could not find any possible functional cataract candidate genes by searching
this region in the current dog genome assembly 2.1
(http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?). Further analyses are
necessary to scan the whole genomic sequence of the PITX3 gene. If no causal
mutations could be detected, identifying of other possible candidate genes for CAT
and investigation of these candidates in the German Pinscher is another approach.
In further molecular genetic analyses, larger samples of closely related dogs
ophthalmologically examined at a young age should be considered. In order to
General discussion
97
achieve this aim, blood samples should be collected in the course of
ophthalmological examinations. In addition, lens tissue of dogs which underwent
cataract surgery should be collected and included in the molecular genetic analyses
to make larger highly informative samples available for molecular genetic analyses.
The candidate gene approach alone may be not sufficient to clarify the molecular
background of primary cataracts in the German Pinscher. For example, to date in
only two dog breeds the molecular genetic cause of CAT could be identified
(Mellersh et al., 2006), although many breeds are affected and a number of scientists
do research on this field. Therefore, other methods than the candidate gene
approach have to be used in breeds where all other known candidate genes can be
excluded as causative for CAT. One approved method is the whole genome scan
using microsatellite markers with an appropriate coverage of the whole chromosome.
The disadvantage of this method is the need for many examined probands with an
informative family structure to find significant results. Since 2007, a commercial SNP
microarray based on the fully sequenced dog genome is available (Lindblah-Toh et
al., 2005). This method offers the opportunity of very fast genotyping of a large
number of SNPs, equally covering the whole dog genome. As the evaluation of the
retrieved data can be based only on association tests, a familial structure of the
proband material is not necessary. The disadvantage here is that the method is not
yet approved in all dog breeds, and therefore an even coverage of the whole genome
with SNP markers may not be warranted in every dog breed.
General discussion
98
References
Beby, F., Morle, L., Michon, L., Edery, P., Burillon, C., et al., 2003. The genetics of
hereditary cataract. Journal français d'ophtalmologie 26, 400-408.
Davidson, M.G., Nelms, S.R., 1999. Diseases of the lens and cataract formation. In:
Veterinary Ophthalmology, 3rd edition, Lippincott/Williams & Wilkins,
Philadelphia.
Graw, J., 2004. Congenital hereditary cataracts. The International journal of
developmental biology 48, 1031-1044.
Helper, L.C., 1989. Magrane´s Canine Ophthalmology. 4th edition, Lea & Febiger,
Philadelphia.
House, A.K., Summerfield, N.J., German, A.J., Noble, P.J.M., Ibbarola, P.,
Brockmann, D.J., 2005. Unususual vascular ring anomaly associated with a
persistent right aortic arch in two dogs. Journal of Small Animal Practice 6, 585-
590.
Lee, M.-L., Chen, H.-N., Chen, M., Tsao, L.-Y., Wang, B.-T., Lee, M.-H., Chiu, I.-S.,
2006. Persistent fifth aortic arch associated with 22q11.2 deletion syndrome.
Journal of the Formosan Medical Association 105, 284-289.
Lepännen, M., Martenson, J., Mäki, K., 2001. Results of ophthalomological screening
examinations of German Pinschers in Finland – a retrospective study. Veterinary
Ophthalmology 4, 165-169.
Lindblah-Toh, K., Wade, C.M., Mikkelsen, T.S., Karlsson, E.K., Jaffe, D.B., et al.,
2005. Genome sequence , comparative analysis and haplotype structure of the
domestic dog. Nature 8, 803-819.
Mellersh, C.S., Pettitt, L., Forman, O.P., Vaudin, M., Barnett, K.C., 2006.
Identification of mutations in HSF4 in dogs of three different breeds with
hereditary cataracts. Veterinary Ophthalmology 9, 369-378.
Patterson, D.F., 1989. Hereditary congenital heart defects in dogs. Journal of Small
Animal Practice 30, 153-165.
Rauch, R., Rauch, A., Koch, A., Zink, S., Kaulitz, R., Girisch, M., Singer, H., Hofbeck,
M., 2004. Laterality of the aortic arch and anomalies of the subclavian artery –
General discussion
99
reliable indicators for 22q11.2 deletion syndromes? European Journal of
Paediatrics 163, 642-645.
Reddy, M.A., Francis, P.J., Berry, V., Bhattacharya, S.S., Moore, A.T., 2004.
Molecular genetic basis of inherited cataract and associated phenotypes. Survey
of Ophthalmology 49, 300-315.
Rubin, L.F., Satterfield, T.S., 1989. Inherited eye diseases in purebreed dogs.
Williams & Wilkins, Baltimore.
Slatter, D., 2001. Fundamentals of Veterinary Ophthalmology, 3rd edition, Saunders,
Philadelphia.
CHAPTER 7
Summary
Summary
102
7 Summary
Julia Menzel (2010)
Population and molecular genetic analysis of persistent right aortic arch and primary cataracts in the German Pinscher The German Pinscher is a dog breed which is affected by primary cataracts (CAT) as
well as persistent right aortic arch (PRAA). The first purpose of the present study was
to describe the occurrence of CAT and a rare form of PRAA in the German Pinscher
population and to analyze the mode of inheritance of both diseases. The second
purpose of the work was to analyze the molecular background of CAT and PRAA this
dog breed.
The present study is the first report of the occurrence of PRAA in the German
Pinscher. Medical records and contrast oesophagrams for 18 dogs of 16 different
litters of the last 10 years were evaluated. Three of them underwent further
investigation at the Hannover Institute for Animal Breeding and Genetics. In all of
these three German Pinschers, PRAA occurs in combination with an aberrant left
subclavian artery and a left ligamentum arteriosum with its origin on the left
subclavian artery (PRAA-SA-LA). The main cause of the constriction of the
oesophagus in all three cases was the aberrant left sublavian artery in combination
with the left ligamentum arteriosum. This combination of anomalies is very rare and
only described in two isolated cases of other dog breeds before. Investigation of the
pedigrees of the affected German Pinschers showed close relationship among all 18
dogs. In addition, these dogs were found to be significant more inbred than a
contemporary group of non-affected individuals. The close relationships and high
inbreeding coefficients suggest that PRAA in German Pinschers is an inherited
defect. We could not determine the mode of inheritance, but following the results of
the simple segregation analysis using the Singles method, a monogenic autosomal
recessive mode of inheritance for this disease in the German Pinscher could be
rejected. A complex, polygenetic mode of inheritance is considered.
Summary
103
The results of comparative genetic research made the canine TBX1 gene a
candidate for PRAA in the German Pinscher. This gene is highly suspicious to be
involved in the pathogenesis of cardiovascular malformations seen in the DiGeorge
syndrome/del22q11.2 syndrome in humans. Comparative mapping of the DiGeorge
region in the dog revealed that this region is mapped to the telomeric end of canine
chromosome 26 (CFA26) and seemed to be conserved in the dog. We genotyped 37
microsatellite markers located within or close to this region on CFA26 in two German
Pinscher families with PRAA-affected dogs and tested them for linkage and
association. A genome-wide significant region was located close to the TBX1 gene.
Sequencing of TBX1 and 13 other genes within the canine DiDeorge region revealed
a total 18 single nucleotide polymorphisms within these genes. Two of them were
found to be associated with the PRAA phenotype in the German Pinscher and
indicated a deletion in this region. A larger number of probands should be used for
further analyses in order to confirm the role of TBX1 in the molecular background of
PRAA in the German Pinscher.
For the population and molecular genetic analyses of CAT in the German Pinscher
population in Germany, a total of 443 eye examination reports of 261 dogs were
analyzed. The examinations were performed by specialized and certified
ophthalmologists using slit-lamp biomicroscopy and indirect ophthalmoscopy. All
results were recorded on official forms based on the standardized eye examination
form corresponding to the European College of Veterinary Ophthalmologists. The
information from the recording forms was linked with the pedigree data of the
German Pinschers. CAT was diagnosed in 15.33% of all examined dogs. The mean
age of onset of CAT was 3.8 ± 1.6 years. A bilateral anterior cortical CAT was found
to be the most prevalent form of CAT in the German Pinscher. Pedigree analysis
revealed a significant higher coefficient of inbreeding as well as a significant higher
coefficient of relationship in the group of CAT-affected German Pinschers than in a
contemporary group of non-affected German Pinschers. The pedigrees supported a
monogenic autosomal recessive inheritance pattern.
Summary
104
The statistical analyses were followed by molecular genetic investigations of CAT.
For this purpose, we searched the literature for genes which are known to be
involved in the pathogenesis of CAT in humans or other animals. We found more
than 30 genes which had to be considered as candidates for primary cataracts. We
genotyped flanking microsatellites for 20 well known CAT-associated genes in CAT-
affected and unaffected German Pinschers and tested them for linkage and
association. For delimitation of a linked region on canine chromosome 28 (CFA28),
genotyping of eight additional microsatellite markers on this chromosome was
performed. We found a genome-wide significant genomic region on CFA28.
Sequencing of the candidate gene PITX3, which is located within the linked region,
revealed no polymorphisms associated with CAT in the German Pinscher.
Sequencing of exon 2 and 3 failed partly despite repeated attempts, the cause could
be the abundance GC. Sequencing of the whole genomic sequence should be
completed in order to scan these regions completely for polymorphisms.
CHAPTER 8
Erweiterte Zusammenfassung
Erweiterte Zusammenfassung
106
8 Erweiterte Zusammenfassung Populationsgenetische und molekulargenetische Analysen des persistierenden rechten Aortenbogens und der primären Katarakt beim Deutschen Pinscher Julia Menzel (2010) 8.1 Einleitung
Der deutsche Pinscher ist eine traditionsreiche Hunderasse, die gemeinsame
Wurzeln mit dem Dobermann Pinscher, dem Zwergpinscher, dem Affenpinscher,
dem Zwergschnauzer, dem Standard Schnauzer und dem Riesenschnauzer hat. Die
ersten Abbildungen der Rasse finden sich in einem Buch aus dem Jahr 1884. Mitte
des 20. Jahrhunderts war die Rasse durch die Folgen der beiden Weltkriege stark
dezimiert und musste vollständig neu aufgebaut werden. Diese Aufgabe übernahm
Werner Jung, indem er das Land nach den wenigen verbliebenen, möglichst
rassetypische Pinschern durchsuchte und diese dann mit vier besonders großen
Zwergpinschern kreuzte. Die meisten Deutschen Pinscher heute sind Nachkommen
dieser wenigen Hunde. Als Folge dessen hat der moderne Deutsche Pinscher einen
relativ kleinen Genpool, eine Tatsache, die besondere Aufmerksamkeit im Hinblick
auf mögliche rassespezifische Erkrankungen verlangt. Die zunehmende Kenntnis
von Prävalenz und Pathogenese von Augen- und Herzerkrankungen innerhalb dieser
Rasse erfordert eine Anpassung und Neuentwicklung von Zuchtrichtlinien, um das
Auftreten von diesen vermutlich erblichen Erkrankungen möglichst stark zu
reduzieren. Deswegen ist es von besonderer Bedeutung, die populationsgenetischen
und molekulargenetischen Hintergründe dieser Erkrankungen aufzuklären.
Gefäßringanomalien („vascular ring anomalies“, VRAs) sind angeborene
Missbildungen der großen Herzstammgefäße und Folge einer Fehlentwicklung der
embryonalen Aortenbögen. Beim Hund ist die häufigste Gefäßringanomalie der
persistierende rechte Aortenbogen („persistent right aortic arch“, PRAA). Bei
erkrankten Hunden wird die Aorta während der embryonalen Entwicklung vom
rechten vierten Aortenbogen anstatt vom linken vierten Aortenbogen gebildet. Die
Erweiterte Zusammenfassung
107
Folge ist eine Einschnürung der Speiseröhre durch den entstehenden Gefäßring, die
zu verschiedenen Symptome wie Regurgitieren von fester Nahrung führt, meist ab
dem Welpenalter. Beim Deutschen Pinscher tritt eine ausgesprochen seltene Form
des persistierenden rechten Aortenbogens auf, die aus einer Kombination
verschiedener Missbildungen zusammengesetzt ist. Zusätzlich zum rechten
Aortenbogen liegt eine retroösophageal verlaufende Arteria subclavia sinistra vor, an
der das Ligamentum arteriosum seinen Ursprung hat.
Als primäre Katarakt (CAT) ist jegliche Trübung der Augenlinse definiert, die nicht im
Zusammenhang mit anderen Augenerkrankungen oder in Folge von systemischen
Erkrankungen auftritt. Es handelt sich dabei um eine bei Rassehunden sehr weit
verbreitete Erkrankung, die weltweit mehr als 120 Hunderassen betrifft. Die
Prävalenz bewegt sich zwischen 1,8 und 88 %, das durchschnittliche Alter bei
erstmaligem Auftreten der Erkrankung ist dabei äußerst variabel. Meist wird ein
erblicher Hintergrund angenommen oder ist bereits belegt, mehrheitlich wurde ein
rezessiver Erbgang nachgewiesen. Die einzige wirksame Behandlung der reifen
Katarakt stellt zum jetzigen Zeitpunkt die Operation dar, was die Bekämpfung der
erblichen Katarakt nicht nur aus Tierschutzgründen, sondern auch aus
wirtschaftlichen Gründen relevant erscheinen lässt. Die meisten
Rassehundezuchtverbände haben bereits Selektionsstrategien entwickelt, um die
Prävalenz von primärer Katarakt in ihrer Zucht zu reduzieren. Nur Hunden, die frei
von erblichen Augenerkrankungen sind, sollte die Zuchterlaubnis erteilt werden. Die
Tatsache, dass erbliche Katarakte beim Hund sich häufig erst nach Erreichen der
Zuchtreife entwickeln, beeinträchtigt die Effektivität dieser Methode. Im Falle eines
rezessiven Erbgangs wird es noch schwerer, das defekte Allel aus der Population zu
entfernen, da es sich über Generationen hinweg unbemerkt innerhalb der Population
verbreiten kann und nur dann erkannt wird, wenn es von beiden Eltern an den
Nachkommen weitergegeben wird. Inzucht erhöht die Wahrscheinlichkeit, dass
dieser Fall eintritt.
Für beide hier untersuchten Erkrankungen wäre ein DNA-Test, der sicher angibt, ob
ein Tier das Defektallel homozygot, heterozygot oder gar nicht in sich trägt, sehr
hilfreich. Zusammen mit einem passenden Zuchtprogramm könnte hiermit die
Erweiterte Zusammenfassung
108
Prävalenz der Erkrankungen viel schneller und effektiver gesenkt werden. Außerdem
könnte so das Risiko einer selektionsbedingten „Flaschenhalsbildung“ minimiert
werden.
Ziel dieser Studie war es, das Auftreten des persistierenden rechten Aortenbogens
und der primären Katarakt beim Deutschen Pinscher zu beschreiben und die
molekulargenetischen Hintergründe zu analysieren.
8.2 Seltene Gefäßringanomalie in Kombination mit einem persistierenden rechten Aortenbogen und einer aberranten Arteria subclavia sinistra beim Deutschen Pinscher Einleitung Ziel dieser Studie war es, das Auftreten einer spezifischen Form des persistierenden
rechten Aortenbogens beim Deutschen Pinscher zu beschreiben und den Erbgang
bei dieser Hunderasse zu analysieren. Die hier zu beschreibende Form des
persistierenden rechten Aortenbogens ist durch das gemeinsame Auftreten mit einer
retroösophageal verlaufenden Arteria subclavia sinistra und einem an dieser Arterie
seinen Ursprung nehmenden Ligamentum arteriosum charakterisiert. Dieser seltene
Verlauf des Ligamentum arteriosum verstärkt zusätzlich die Kompression der
Speiseröhre durch den Gefäßring und wurde bisher erst bei zwei Einzelfällen andere
Hunderassen beschrieben.
Bei den betroffenen neugeborenen Welpen sind zunächst keine Symptome zu
erkennen. Diese treten in der Regel erst dann auf, wenn die Welpen erstmalig feste
Nahrung zu sich nehmen; typisch ist dann das Regurgitieren nach der
Futteraufnahme. Durch die Einschnürung der Speiseröhre auf Höhe der Herzbasis ist
ein Weitertransport der Ingesta nicht möglich oder zumindest erheblich
beeinträchtigt, was zur Regurgitation führt. Zusätzlich verbleiben Teile des
aufgenommenen Futters kranial der Konstriktion in der Speiseröhre und verursachen
so auf Dauer eine Ösophagusdilatation im präkardialen Bereich. Betroffene Welpen
bleiben im Wachstum zurück und verlieren bei unvermindertem oder sogar
Erweiterte Zusammenfassung
109
gesteigertem Appetit an Gewicht. Auf Röntgenaufnahmen des Thorax nach oraler
Verabreichung von Kontrastmitteln lässt sich die Speiseröhrendilatation kranial des
Herzen darstellen, die eingeschnürte Stelle befindet sich dabei auf Höhe der
Herzbasis.
Bisher gibt es in der Literatur keine Berichte über das Auftreten des persistierenden
rechten Aortenbogens beim Deutschen Pinscher. Dennoch wurde in den
vergangenen sechs Monaten die hier beschriebene, seltene Form des
persistierenden rechten Aortenbogens bei drei Welpen der Rasse Deutscher
Pinscher diagnostiziert. Weitere fünfzehn Fälle wurden von Züchtern oder Tierärzten
dokumentiert und sind daher teilweise als Verdachtsdiagnose anzusehen.
Material und Methoden Bei allen drei an der Tierärztlichen Hochschule Hannover untersuchten Welpen war
die Ursache der Ösophaguskonstriktion ein persistierender rechter Aortenbogen,
eine retroösophageal verlaufende linke Arteria subclavia und ein linkes Ligamentum
arteriosum, das seinen Ursprung an der Arteria subclavia hatte. Bei den anderen
fünfzehn Fällen handelt es sich um Berichte von Tierärzten und Züchtern aus
Deutschland und den Niederlanden. Einer dieser fünfzehn Welpen wurde in einem
privaten Institut für Pathologie in den Niederlanden mit der gleichen selten Diagnose
wie bei den drei in Hannover untersuchten Welpen obduziert. Ein andere Welpe
wurde in München operiert und lebt nun schon mehrere Jahre ohne Probleme bei
seinem Besitzer. Bei einer inzwischen sieben Jahre alten Hündin wurde im Alter von
acht Monaten die Verdachtsdiagnose persistierender rechter Aortenbogen gestellt.
Diese Hündin erfährt seitdem ein spezielles Fütterungsmanagement und lebt ohne
größere Beeinträchtigungen mit der Erkrankung.
Die Abstammungsdaten der erkrankten Hunde wurden uns vom Pinscher-
Schnauzer-Klub e.V. (PSK) zur Verfügung gestellt und wurden unter anderem zur
einfachen Segregationsanalyse nach der Singles Methode verwendet, um
herauszufinden, ob die Daten mit einem rezessiven Erbgang kompatibel sind.
Der mittlere Verwandtschaftskoeffizient wurde sowohl für die Gruppe von Deutschen
Pinschern mit betroffenen Nachkommen als auch für eine Vergleichsgruppe
Erweiterte Zusammenfassung
110
berechnet. Der mittlere Inzuchtkoeffizient der Gruppe von erkrankten Hunden wurde
ebenfalls berechnet und dem mittleren Inzuchtkoeffizienten einer Vergleichsgruppe
gegenübergestellt. Für die Berechnungen wurden Abstammungsdaten über acht
Generationen verwendet, dabei betrug die Vollständigkeit der Daten mehr als 95%.
Ergebnisse Bei dem ersten in Hannover untersuchten Tier, einem drei Wochen alten weiblichen
Welpen der Rasse Deutscher Pinscher, wurde nach pathologisch-anatomischer
Untersuchung ein persistierender rechter Aortenbogen in Kombination mit einer
aberranten linken Arteria subclavia und einem linken Ligamentum arteriosum
festgestellt. Das Ligamentum arteriosum hatte seinen Ursprung an der linken Arteria
subclavia. Als Hauptgrund der Speiseröhrenkonstriktion konnte die aberrante Arteria
subclavia in Kombination mit dem Ligamentum arteriosum ermittelt werden, der
Aortenbogen trug nur zu einem kleineren Anteil zu der Einschnürung bei. Der Welpe
war zuvor vom Haustierarzt wegen andauerndem Regurgitieren und schlechter
allgemeiner körperlicher Verfassung eingeschläfert worden. Bei den anderen beiden
in Hannover untersuchten Tieren handelte es sich um zwei Welpen aus einem Wurf
eines niederländischen Züchters. Beide Welpen wurden in der Klinik für Kleintiere
der Tierärztlichen Hochschule Hannover vollständig untersucht. Bei dem einen Tier
(Welpe 2) wurde zusätzlich zur hochgradigen Dilatation der Speiseröhre noch ein
Ventrikelseptumdefekt festgestellt. Aufgrund der schlechten Prognose wurde er
eingeschläfert und zur pathologisch-anatomischen Untersuchung übersendet. Bei
dem anderen Welpen (Welpe 3) war die Dilatation der Speiseröhre weniger
ausgeprägt und die körperliche Verfassung insgesamt besser, daher wurde dieser
Welpe am nächsten Tag operiert. Nach Eröffnung der Brusthöhle ergab sich fast ein
identisches Bild wie bei der pathologisch-anatomischen Untersuchung von Welpe 1.
Das Ligamentum arteriosum wurde ligiert und anschließend durchtrennt. Danach war
keine Kompression der Speiseröhre mehr zu erkennen, die Arteria subclavia wurde
daher nicht weiter manipuliert. Dem Welpen ging es bereits am Tag nach der
Operation gut; er erhielt für die Dauer von 3 Monaten ein spezielles
Fütterungsmanagement und lebt inzwischen ohne gesundheitliche Einschränkungen
Erweiterte Zusammenfassung
111
bei seinen neuen Besitzern. Die Befunde der pathologisch-anatomischen
Untersuchung von Welpe 3 waren fast identisch mit den Befunden von Welpe 1. Die
Dilatation der Speiseröhre war bei diesem Tier noch stärker ausgeprägt als bei den
anderen beiden Welpen, im präkardialen Bereich war der Durchmesser des
Oesophagus etwa fünfmal größer als auf Höhe der Herzbasis.
Die Verwandtschaftsbeziehungen aller erkrankten Tiere konnten in einem Pedigree
dargestellt werden. Verwandtschafts- und Inzuchtkoeffizienten waren bei der Gruppe
von Erkrankten signifikant höher als in einer Vergleichsgruppe. Alle betroffenen
Welpen hatten nicht betroffene Eltern, die Anzahl betroffener weiblicher und
betroffener männlicher Tiere war nahezu identisch. Deswegen konnte ein X–
chromosomaler Erbgang genauso wie ein monogen autosomal dominanter Erbgang
und ein mitochondrialer Erbgang ausgeschlossen werden. Die Ergebnisse der
einfachen Segregationsanalysen nach der Singles-Methode wiesen nicht auf einen
monogen autosomal rezessiven Erbgang hin.
Diskussion Die retroösophageale Arteria subclavia sinistra in Kombination mit dem linken
Ligamentum arteriosum mit seinem Ursprung an der Arteria subclavia sinistra war in
allen drei untersuchten Fällen die Hauptursache der Ösophaguskonstriktion. Diese
Kombination von Anomalien ähnelt stark einer erblichen Anomalie des Menschen,
bei der zusätzlich ein Divertikulum am Ursprung der Arteria subclavia sinistra auftritt
(Kommerell`s diverticulum). Die einzige dauerhaft Erfolg versprechende Therapie
von Gefäßringanomalien ist die chirurgische Korrektur. Medikamentelle Therapie in
Kombination mit einem angepassten Fütterungsmanagement wird als nicht effektiv
genug angesehen und führt in der Regel langfristig zur Verschlechterung der
Situation. Trotzdem lebt einer der betroffenen Deutschen Pinscher seit 7 Jahren
ohne ernsthafte Beeinträchtigungen mit der Erkrankung.
Zuchtstudien beim Deutschen Schäferhund sowie epidemiologische Studien beim
Deutschen Schäferhund und beim Irish Setter in der Vergangenheit haben gezeigt,
dass diese beiden Rassen eine Prädisposition für die Entwicklung eines
Erweiterte Zusammenfassung
112
persistierenden rechten Aortenbogen aufweisen, der Erbgang ist vermutlich komplex
und polygen.
Die engen verwandtschaftlichen Beziehungen und hohen Inzuchtkoeffizienten legen
nahe, dass der persistierende rechte Aortenbogen beim Deutschen Pinscher ein
erblicher Defekt ist. Je höher der Inzuchtkoeffizient ist, desto wahrscheinlicher
manifestiert sich ein rezessiver Defekt bei den Nachkommen. Da ein monogen
autosomal rezessiver Erbgang mit hoher Wahrscheinlichkeit ausgeschlossen werden
konnte, scheint ein oligo- oder polygener Erbgang oder der Beitrag mehrerer
Mutationen einer Genregion am wahrscheinlichsten.
Vergleichende genetische Studien ergaben, dass Anomalien der Herzstammgefäße
beim Menschen oft im Zusammenhang mit einer Deletion der Chromosom 22q11.2 -
Region stehen, die auch als DiGeorge-Region bekannt ist. Monosomie 22q11.2
wurde bei 46% aller Patienten mit einem persistierenden rechten Aortenbogen
festgestellt, aber nur bei 30% der Patienten mit einem linken Aortenbogen. Die
Mikrodeletion wurde außerdem bei 81% der Patienten mit, hingegen nur bei 17% der
Patienten ohne Anomalien der Arteria subclavia festgestellt. Die Inzidenz von
Monosomie 22q11.2 war also wesentlich stärker mit dem Vorliegen von Anomalien
der Arteria subclavia als mit der Seitigkeit des Aortenbogen assoziiert. Studien mit
Mäusen ergaben, dass eines der Gene innerhalb der DiGeorge - Region, TBX1, ein
aussichtsreiches Kandidatengen für die kardiovaskulären Missbildungen im
Zusammenhang mit dem DiGeorge-Syndrom beim Menschen ist. Die DiGeorge-
Region ist auch beim Hund vorhanden und ist bei dieser Spezies auf Chromosom 26
lokalisiert.
Schlussfolgerungen Monosomie 22q11.2 ist stark mit dem Vorliegen von Anomalien der Arteria subclavia
assoziiert, die bei allen drei hier untersuchten Welpen gefunden wurden. Eine
Kopplungsanalyse von Genorten im Bereich der DiGeorge-Region mit dem
persistierenden rechten Aortenbogen beim Deutschen Pinscher könnte klären, ob
Mutationen in dieser Region verantwortlich für das Auftreten der Missbildung sind.
Erweiterte Zusammenfassung
113
8.3 Evaluierung des TBX1 – Gens als Kandidatengen für eine seltene Form des persistierenden rechten Aortenbogens beim Deutschen Pinscher Einleitung Der persistierende rechte Aortenbogen ist die häufigste angeborene
Gefäßringanomalie und bei einigen Hunderasen wie dem Deutschen Schäferhund
und dem Irish Setter verbreitet. Beim deutschen Pinscher tritt eine seltene Form des
persistierenden rechten Aortenbogens auf, die mit einer aberranten Arteria subclavia
sinistra und einem Ligamentum arteriosum assoziiert ist, das von der Arteria
pulomonalis zur aberranten Arteria subclavia sinistra verläuft anstatt zum
Aortenbogen. Diese zusätzliche Missbildung verstärkt den Druck auf die
Speiseröhre. Vergleichende genetische Studien ergaben, dass TBX1 ein
aussichtsreiches Kandidatengen für ähnliche Anomalien beim Menschen und bei der
Maus darstellt, die dort im Zusammenhang mit dem DiGeorge-Syndrom auftreten.
Das Gen TBX1 gehört zur Familie der T-Box Gene, die eine wichtige Rolle bei der
Regulation von Entwicklungsprozessen spielen, unter anderem bei der Angiogenese,
Festlegung der Rechts-Links-Symmetrie, Herzentwicklung und
Mesodermentwicklung. Das Gen ist auch beim Hund konserviert. In der vorliegenden
Studie evaluierten wir die genomische Struktur des TBX1 - Gens beim Hund und
untersuchten die vollständige genomische Sequenz von TBX1 sowie Teile der Exons
mit flankierenden intronischen Regionen von 13 anderen Genen innerhalb der
caninen DiGeorge-Region auf Polymorphismen, die für Kopplungs- und
Assoziationsanalysen mit dem PRAA-Phänotyp beim Deutschen Pinscher genutzt
werden können.
Material und Methoden Vom Pinscher-Schnauzer-Klub 1895 e.V. wurden für alle Hunde Abstammungsdaten
zur Verfügung gestellt. Wir wählten insgesamt 45 Hunde aus zwei verschiedenen
Familien aus, darunter drei von der gleichen, seltenen Form des persistierenden
rechten Aortenbogens betroffene Tiere. Nach den ersten Ergebnissen der
Kopplungs- und Assoziationsanalysen wurden alle verfügbaren Vollgeschwister
Erweiterte Zusammenfassung
114
dieser betroffenen Tiere als Anlageträger gekennzeichnet, um aussagekräftigere
Ergebnisse zu erzielen. Außerdem wurden drei nicht erkrankte Tiere anderer Rassen
als Kontrolltiere mit untersucht. Zwei Milliliter EDTA-Blut wurde von jedem Tier
gesammelt, aus dem dann die DNA isoliert wurde.
Es wurden insgesamt 37 Mikrosatelliten auf Chromosom 26 für jedes Tier typisiert.
Die nicht-parametrische Kopplungsanalyse wurde basierend auf dem identical-by-
descent-Verfahren unter Verwendung der Software MERLIN 1.1.2 durchgeführt.
Dabei wurden die Markerallele auf Kosegregation mit der phänotypischen
Ausprägung des persistierenden rechten Aortenbogens beim Deutschen Pinscher
getestet. Die Daten wurden mit dem Softwarepaket SAS bearbeitet, um die Marker
bezüglich Allelfrequenzen, Heterozygotiegrad und Polymorphismus-
Informationsgehalt charakterisieren zu können. Für den Assoziationstest wurde eine
case-control Analyse basierend auf χ2-Tests für Genotypen und Allele durchgeführt.
Außerdem wurde eine Sequenz- und Mutationsanalyse für insgesamt 14 Gene auf
Chromosom 26 bei 11 Tieren durchgeführt, deren Ergebnisse in die Kopplungs- und
Assoziationsanalysen mit einbezogen wurden.
Ergebnisse und Diskussion
Mittels der nicht-parametrischen Kopplungsanalyse der Mikrosatelliten konnte eine
signifikante Kopplung der Marker 26_31.48 und 26_31.99 mit dem Phänotyp des
persistierenden rechten Aortenbogens beim Deutschen Pinscher festgestellt werden.
Mittel Sequenz- und Mutationsanalyse der Gene innerhalb der DiGeorge-Region
konnten insgesamt 18 SNPs (single nucloetide polymorphisms) für die Gene GNBL1,
TXNRD2, CDC45L, UFD1, MRPL40, CLTCL1, SLC25A1 und GSC2 gefunden
werden. Zwei dieser SNPs im Bereich des Kandidatengens TBX1 zeigten eine
signifikante Kopplung mit dem Phänotyp des persistierenden rechten Aortenbogens
beim Deutschen Pinscher und wiesen auf eine Deletion in diesem Bereich bei den
betroffenen Tieren hin. Um die Bedeutung dieses Gens als ursächlich für den
persistierenden rechten Aortenbogen zu verifizieren, sind weitere Untersuchungen
an einer größeren Anzahl betroffener und nicht betroffener Tiere nötig.
Erweiterte Zusammenfassung
115
8.4 Prävalenz und Auftreten der primären Katarakt in der Deutschen Pinscher Population in Deutschland Einleitung Primäre Katarakt (CAT) ist eine der häufigsten Ursachen für visuelle
Beeinträchtigungen und Blindheit bei Rassehunden, weltweit sind mehr als 120
Hunderassen betroffen. Bei vielen dieser Rassen konnte eine Erblichkeit der
primären Katarakt nachgewiesen werden. Mehrheitlich wird ein rezessiver Erbgang
angenommen, aber auch dominante Formen sind beschrieben. In der bisher einzigen
Veröffentlichung zur primären Katarakt beim Deutschen Pinscher, einer Studie aus
Finnland, wird ein unvollständig dominanter Erbgang vermutet
Da bisher ein kostenintensiver chirurgischer Eingriff die einzige effektive Therapie
der Erkrankung darstellt, hat die Vermeidung des Auftretens besondere Bedeutung
für das Wohlergehen der Tiere und auch aus wirtschaftlichen Gesichtspunkten. Viele
Zuchtverbände haben aus diesem Grund regelmäßige verpflichtende
Augenuntersuchungen zur Bedingung für die Zuchtzulassung gemacht. Oft
entwickelt sich eine Katarakt aber erst in einem Alter, in die meisten Tiere ihre ersten
Zuchteinsätze bereits hinter sich haben, daher ist diese Methode nur begrenzt
erfolgreich. Ein rezessiver Erbgang erschwert das Vorgehen zusätzlich.
Da beim Deutschen Pinscher eine Prädisposition für primäre Katarakt vorliegt,
besteht Grund zur Annnahme, das es sich um eine erbliche Erkrankung handelt. Ziel
dieser Studie war es, die Prävalenz und das Auftreten der primären Katarakt in der
Deutschen Pinscher Population zu charakterisieren und den Erbgang bei dieser
Hunderasse zu analysieren.
Material und Methoden
Der Pinscher-Schnauzer-Klub 1895 e.V. (PSK) und der Dortmunder Kreis (DOK)
stellten die Daten für die vorliegende Arbeit zur Verfügung. Die Studie basiert auf
Untersuchungsbögen für erbliche Augenerkrankungen von 261 Deutschen
Pinschern, die Daten wurden zwischen Januar 1995 und Oktober 2009 erhoben. Die
ophthalmologischen Untersuchungen wurden von spezialisierten und DOK-
Erweiterte Zusammenfassung
116
zertifizierten Tierärzten durchgeführt. Insgesamt wurden 443 Untersuchungsbögen
von 261 verschiedenen Hunden ausgewertet; die Untersuchungen wurden von 61
verschiedenen Gutachtern durchgeführt. Die Informationen der Untersuchungsbögen
wurden zusammen mit den Pedigreedaten ausgewertet, die uns vom PSK zur
Verfügung gestellt wurden.
Die Pedigreedaten wurden für eine einfache Segregationsanalyse nach der Singles-
Methode genutzt, um zu testen ob die Daten mit einem relativ einfachen
Mendel’schen Modell, einem rezessiven Erbgang, kompatibel sind. Verwandtschafts-
und Inzuchtkoeffizienten wurden für betroffene Tiere und eine Vergleichsgruppe
kalkuliert, dabei wurden Abstammungsinformationen über 8 Generationen
berücksichtigt. Die Vollständigkeit der Pedigrees lag jeweils über 95%.
Ergebnisse Bei 50 Untersuchungen von 40 verschiedenen Hunden (15,33% aller Hunde) wurde
CAT diagnostiziert. Sieben dieser 40 Hunde wurden noch weitere male untersucht,
nachdem die Diagnose CAT das erste Mal gestellt wurde. Bei 67% der betroffenen
Hunde wurde bei der Erstuntersuchung CAT diagnostiziert. Bei 22,50% der
betroffenen Tiere trat eine einseitige Katarakt auf, bei 62,50% eine beidseitige
Katarakt. CAT wurde bei 55% der betroffenen Tiere als „anterior kortikal“
beschrieben, bei 15% als „posterior polar“, bei 2.5% als „nuklear“, bei 10% als
„anterior kortikal und posterior polar“ und bei 7,5% als „posterior polar und nuklear“,
während bei vier Tieren die Angaben fehlten. Das durchschnittliche Alter bei
Feststellung von CAT lag bei 3,8 ± 1,6 Jahren. Das jüngste der betroffenen Tiere war
0,34 Jahre alt, das älteste 7,44 Jahre als die Erstdiagnose CAT gestellt wurde. Fast
60% der betroffenen Tiere waren bei der Erstdiagnose weniger als vier Jahre alt. Bei
13.68% der männlichen Tiere und bei 16.67% der weiblichen Tiere wurde CAT
diagnostiziert.
Die meisten Hunde wurden nur einmal untersucht, 20,69% wurden zweimal
untersucht und 18,39% wurden dreimal oder häufiger untersucht. Das Alter zum
Zeitpunkt der Untersuchung lag zwischen 0,34 und 13,77 Jahren. Die meisten Tiere
wurden im Alter von zwei bis fünf Jahren untersucht. Die Erstuntersuchung erfolgte
Erweiterte Zusammenfassung
117
im Durchschnitt im Alter von 3,0 ± 2,0 Jahren, die letzte Untersuchung bei mehrfach
untersuchten Tieren erfolgte durchschnittlich im Alter von 4,0 ± 2,3 Jahren. 25% aller
Tiere wurden mindestens einmal im alter von über 5 Jahren untersucht. Die Anzahl
der weiblichen (55,56%) und männlichen (44,44%) untersuchten Tiere wies keinen
signifikanten Unterschied auf. Die untersuchten Hunde stammten aus 83
verschiedenen Zwingern.
Die Analyse der Abstammungsdaten zeigte die engen verwandtschaftlichen
Verhältnisse der von CAT betroffenen Tiere auf. Bis auf zwei Tiere konnten alle
betroffenen Hunde in einem Pedigree dargestellt werden. CAT trat nicht in jeder
Generation auf, was ein Hinweis auf eine rezessive Vererbung ist. Der
durchschnittliche Verwandschaftskoeffizient sowie der durchschnittliche
Inzuchtkoeffizient waren bei der Gruppe von CAT-betroffenen Tieren signifikant
höher als bei einer Vergleichsgruppe von nicht betroffenen Deutschen Pinschern. Die
Ergebnisse der einfachen Segregationsanalyse nach der Singles-Methode zeigten,
dass die beobachtete Verteilung mit einem einfachen rezessiven Erbgang und einer
Segregationsfrequenz von p = 0,303 konsistent ist.
Diskussion Da die Prävalenz von CAT in der Deutschen Pinscher Population in Deutschland
signifikant höher ist als bei einer Vielzahl von anderen Hunderassen in Deutschland,
handelt es sich mit großer Wahrscheinlichkeit um eine erbliche Erkrankung bei dieser
Rasse. Im Vergleich mit einer finnischen Studie zu erblichen Augenerkrankungen in
der Deutschen Pinscher Population in Finnland konnten wir in der vorliegende Studie
eine deutlich höhere Prävalenz von CAT und ein wesentlich niedrigeres Auftrittsalter
der Erkrankung feststellen. Auch die häufigste Lokalisation der Linsentrübung war
unterschiedlich. Daraus lässt sich schließen, dass hier zwei verschieden Arten von
Katarakt segregieren.
Inzuchtkoeffizienten haben nachgewiesenermaßen einen signifikanten Einfluss auf
die Prävalenz von CAT, z.B. beim Englischen Cocker Spaniel. Gerade beim
Vorliegen eines rezessiven Erbgangs erhöht Inzucht die Wahrscheinlichkeit für die
Manifestation einer Erkrankung erheblich. Das hohe Inzuchtlevel in der Deutschen
Erweiterte Zusammenfassung
118
Pinscher Population führt zu einer Akkumulation von Defektallelen in einer
steigenden Anzahl von Zuchttieren.
Das Problem der meisten genetischen Studien zur Untersuchung des Erbgangs von
CAT ist die eingeschränkte Anzahl an untersuchten Anpaarungen und Nachkommen.
Oft manifestiert sich CAT erst im fortgeschrittenen Alter und es Bedarf daher viele
Jahre der Beobachtung. In einigen Studien konnten Vermutungen zum Erbgang
basierend auf visueller Inspektion der verfügbaren Pedigrees geäußert werden. In
der vorliegenden Studie erlaubte die Verteilung der ophthalmologisch untersuchten
Tiere eine einfache Segregationsanalyse. Die Ergebnisse dieser Analyse schließen
jedoch andere, komplexere Erbgänge nicht völlig aus.
Das meist späte Auftreten der ersten Anzeichen von CAT stellt ein großes Problem
für Züchter dar, weil viele Tiere ihren ersten Zuchteinsatz bereits hinter sich haben,
wenn die Diagnose CAT gestellt wird. Regelmäßige ophthalmologische
Untersuchungen durch spezialisierte Tierärzte sollten verpflichtend für die Erlaubnis
zum Zuchteinsatz sein, um das Risiko der Vererbung zu reduzieren und die
Prävalenz in der gesamten Zucht zu verringern. Diese Untersuchungen sollten
mindestens jährlich durchgeführt werden, solange die Tiere im Zuchteinsatz sind.
Erkrankte Hunde dürfen keinesfalls zur Zucht eingesetzt werden, und es sollte
bevorzugt mit Hunden gezüchtet werden, die alt genug sind, CAT mit großer
Wahrscheinlichkeit ausschließen zu können.
8.5 Evaluierung von 20 Kandidatengenen für primäre Katarakt beim
Deutschen Pinscher Einleitung Primäre Katarakt ist eine häufig auftretende Erkrankung bei Rassehunden, über 120
Rassen weltweit sind betroffenen. Die Erblichkeit von nicht kongenitaler primärer
Katarakt (CAT) wurde bereits bei vielen Rassen nachgewiesen. Der Deutsche
Pinscher ist ebenfalls eine Rasse mit Prädisposition für die Entwicklung einer
primären Katarakt, daher wird angenommen, dass die Erkrankung auch hier einen
Erweiterte Zusammenfassung
119
genetischen Hintergrund besitzt. Die Prävalenz von CAT in der Deutschen Pinscher
Population in Deutschland wurde in einer anderen Studie auf 15,33% geschätzt. Die
Mehrheit der Deutschen Pinscher entwickelt eine beidseitige Katarakt, die am
Häufigsten im vorderen kortikalen Teil der Linse lokalisiert ist. Das durchschnittliche
Alter bei der Erstdiagnose von CAT liegt bei den hier untersuchten Deutschen
Pinschern bei 3,8 Jahren. Aufgrund des relativ späten Manifestationsalters und des
vermutlich rezessiven Erbgangs ist es schwierig Merkmalsträger rechtzeitig zu
erkennen und von der Zucht auszuschließen sowie Anlageträger zu erkennen. Ein
DNA-Test, der zeigt ob ein Tier homozygot für eine CAT verursachende Mutation
oder eine heterozygoter Anlageträger oder frei von CAT verursachenden Mutationen
ist, wäre sehr hilfreich und könnte in Kombination mit einem effektiven
Zuchtprogramm die Prävalenz von CAT beim Deutschen Pinscher erheblich senken.
Heute sind über 20 Gene beim Hund bekannt, die als potentielle Kandidatengene für
CAT angesehen werden, da sie beim Menschen oder bei der Maus mit dieser
Erkrankung assoziiert sind. In der vorliegenden Studie wurden die 20
Kandidatengene nach Mellersh et al. (2006) untersucht.
Material und Methoden Die Ergebnisse ophthalmologischer Untersuchungen von Deutschen Pinschern
wurden vom Dortmunder Kreis (DOK) zur Verfügung gestellt. Alle Untersuchungen
wurden von spezialisierten und zertifizierten Tierärzten basierend auf einem
standardisierten Untersuchungsprotokoll durchgeführt. Nur primäre Katarakte wurden
dabei berücksichtigt. Der Pinscher-Schnauzer-Klub 1895 e.V. (PSK) stellte die
Abstammungsdaten für alle Tiere zur Verfügung. Wir wählten insgesamt 45 Hunde
aus fünf verschiedenen Familien aus, darunter 15 von CAT betroffene Tiere. Bei den
meisten dieser erkrankten Hunde (86,67%) war die Trübung im vorderen kortikalen
Bereich der Linse lokalisiert. Bei 11 Tieren (73,33%) waren beide Augen betroffen,
während bei den restlichen vier Hunden nur das rechte oder nur das linke Auge
betroffen waren. Die Mehrheit der Probanden (70%) wurde zweimal oder dreimal
ophthalmologisch untersucht. Mindestens ein nicht betroffenes Tier aus jeder Familie
wurde ebenfalls untersucht. Nicht betroffene Tiere, bei denen die letzte
Erweiterte Zusammenfassung
120
ophthalmologische Untersuchung im Alter unter 4,5 Jahren erfolgte, wurden als
Hunde mit unbekanntem Phänotyp klassifiziert. zusätzlich testeten wir drei nicht an
CAT erkrankte Hunde anderer Rassen als Kontrolltiere. Zwei Milliliter EDTA-Blut
wurde von jedem Tier gesammelt, aus dem dann die DNA isoliert wurde.
Zur Untersuchung der 20 Kandidatengene verwendeten wir die 36 Mikrosatelliten
sowie PCR-Bedingungen nach Mellersh et al. (2006). Zur Feinkartierung der
gekoppelten Region auf Chromosom 28 wählten wir acht weitere Mikrosatelliten aus.
Alle Mikrosatelliten wurden für alle 45 untersuchten Tiere typisiert. Eine nicht-
parametrische Kopplungsanalyse wurde basierend auf dem identical-by-descent
Verfahren unter Verwendung der Software MERLIN 1.1.2 durchgeführt. Die
Markerallele wurden dabei auf Kosegregation mit dem CAT-Phänotyp beim
Deutschen Pinscher getestet. Alle Marker wurden bezüglich Allelfrequenzen,
Heterozygotiegrad und Polymorphismus-Informationsgehalt charakterisiert. Für den
Assoziationstest wurde eine case-control Analyse basierend auf χ2-Tests für
Genotypen und Allele durchgeführt. Außerdem wurde eine Sequenz- und
Mutationsanalyse für das Gen PITX3 bei 10 Tieren durchgeführt, deren Ergebnisse
ebenfalls in die Kopplungs- und Assoziationsanalysen mit einbezogen wurden.
Ergebnisse und Diskussion Mittels der nicht-parametrischen Kopplungsanalyse der Mikrosatelliten konnte eine
signifikante Kopplung der Marker CF28_16.26, PITX3_28_16.97 und CF28_17.89
mit dem CAT-Phänotyp beim Deutschen Pinscher festgestellt werden. Diese Marker
sind alle auf Chromosom 28 in unmittelbarer Nähe des PITX3-Genes lokalisiert.
Mittels Sequenz- und Mutationsanalysen von PITX3 konnten insgesamt sieben
Zwischenrasse-SNPs gefunden werden, von denen fünf in Exon 3 lokalisiert waren.
Die anderen zwei lagen im Bereich von Intron 2. Es konnten keine Polymorphismen
innerhalb der Rasse Deutscher Pinscher gefunden werden. Die Sequenzierung von
Exon 2 und 3 erfolgte trotz mehrmaligen Versuchen mit unterschiedlichen
Bedingungen nur unvollständig, so dass diese Teile des Gens nur teilweise
untersucht werden konnten. Die Ursache dafür könnte im GC-Reichtum der
untersuchten Sequenzen liegen. Die Sequenzierung der vollständigen genomische
Erweiterte Zusammenfassung
121
Sequenz von PITX3 sollte vorgenommen werden, um möglicherweise
Polymorphismen zu finden, die mit dem CAT-Phänotyp beim Deutschen Pinscher
gekoppelt sind.
Auch die Analyse von cDNA des PITX3-Gens wäre ein weitere Ansatz. Hierfür
müsste allerdings Linsengewebe von erkrankten Tieren gesammelt werden, das
momentan leider nicht zur Verfügung steht.
CHAPTER 9
Appendix
Appendix
124
Laboratory paraphernalia: A . Equipment
A.1. Thermocycler
PCT-100TM Programmable Thermal Controller (MJ Research, Watertown, USA)
PCT-100TM Peltier Thermal Cycler (MJ Research, Watertown, USA)
PCT-200TM Peltier Thermal Cycler (MJ Research, Watertown, USA)
Biometra Professional Thermocycler (Biometra, Göttingen, Germany)
A.2. Automated sequencers
LI-COR Gene Read IR 4200 DNA Analyzer (LI-COR Inc., Lincoln, NE, USA)
LI-COR Gene Read IR 4300 DNA Analyzer (LI-COR Inc., Lincoln, NE, USA)
MegaBACE 500 (Amersham Biosciences, Freiburg, Germany)
A.3. Centrifuges
Sigma centrifuge 4-15 (Sigma Laborzentrifugen GmbH, Osterode, Germany)
Desk-centrifuge 5415D (Eppendorf, Hamburg, Germany)
Speed Vac® Plus (Savant Instruments, Farmingdale, NY, USA)
A.4. Agarose gel electrophoresis and pulsed field gel electrophoresis
Electrophoresis chambers OWL Separation Systems, Portsmouth, NH, USA
Biometra, Göttingen, Germany
BioRad, München, Germany
Generators 2301 Macrodrive 1 (LKB, Bromma, Sweden)
Gel document system BioDocAnalyze 312 nm, Göttingen, Germany
A.5. Pipettes
Multipipette® plus (Eppendorf AG, Hamburg, Germany)
pipetus®-akku (Hirschmann® Laborgeräte GmbH & Co.KG, Eberstadt, Germany)
Appendix
125
Pipetman® (P2, P10, P100, P200, P1000) (Gilson Medical Electronics S.A., Villiers-
le-bel, France)
Pipettor®, Multi 12 Channel (0.1 – 10 µl) (Micronic® systems, Lelystadt, The
Netherlands)
12 Channel Manual Pipettor (0.5 – 10 µl) (Matrix Technologies Corporation,
Cheshire, UK)
12 Channel Manual Pipettor (25 – 200 µl) (Matrix Technologies Corporation,
Cheshire, UK)
8-channel gel loading syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland)
A.6. Others
Milli-Q biocel water purification systems (Millipore GmbH, Eschborn, Germany)
Incubator VT 5042 (Hereaus, Osterode, Germany)
UV-Illuminator 312 nm (Bachhofer, Reutlingen, Germany)
B. Kits
B.1. Isolation of DNA
QIAamp 96 DNA Blood Kit (QIAGEN, Hilden, Germany)
NucleoSpin Kit 96 Blood Qiuck Pure Kit (Macherey-Nagel, Düren, Germany)
B.2. DNA amplification
GenomiPhi TM DNA Amplification Kit (GE Healthcare, Freiburg, Germany)
B.3. Preservation of DNA
RNAlater Solution (Qiagen, Hilden, Germany)
B.4. Isolation of RNA
Nucleospin RNA II-Kit (Macherey-Nagel, Düren, Germany)
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126
B.5. DNA purification
Montagne PCR96 Cleanup Kit (Millipore GmbH, Eschborn, Germany)
MinEluteR 96 UF Plate (QIAGEN, Hilden, Germany)
AutoSeqTM 96 Plate (GE Healthcare, Freiburg, Germany)
B.6. Sequencing
DYEnamic-ET-Terminator Cycle Sequencing Kit (GE Healthcare, Freiburg, Germany)
C. Size standards
100 bp Ladder (New England Biolabs, Schwalbach Taunus, Germany)
1 kb Ladder (New England Biolabs, Schwalbach Taunus, Germany)
IRDyeTM 700 (LI-COR Inc., Lincoln, NE, USA)
IRDyeTM 800 (LI-COR Inc., Lincoln, NE, USA)
D. Reagents and buffers
D.1. APS solution
1 g APS
10 ml H2O
D.2. Bromophenol blue solution
0.5 g bromophenol blue
10 ml 0.5 M EDTA solution
H2O ad 50 ml
D.3. dNTP solution
100 µl dATP [100mM]
100 µl dCTP [100mM]
100 µl dGTP [100mM]
100 µl dTTP [100mM]
Appendix
127
1600 µl H2O
The concentration of each dNTP in the ready-to-use solution is 5 mM
D.4. Gel solution (4%)
13.5 ml Urea/TBE solution (4%)
1.5 ml Rotiphorese® Gel 40 (38% acrylamide and 2% bisacrylamide)
95 µl APS solution (10%)
9.5 µl TEMED
D.5. Gel solution (6%)
12.75 ml Urea/TBE solution (6%)
2.25 ml Rotiphorese® Gel 40 (38% acrylamide and 2% bisacrylamide)
95 µl APS solution (10%)
9.5 µl TEMED
D.6. Loading buffer for agarose gels
EDTA, pH 8 100 mM
Ficoll 400 20% (w/v)
Bromophenol blue 0.25% (w/v)
Xylencyanol 0.25% (w/v)
D.7. Loading buffer for gel electrophoresis
2 ml bromophenol blue solution
20 ml formamide
D.8. TBE-buffer (1x)
100 ml TBE-buffer (10x)
900 ml H2O
D.9. TBE-buffer (10x)
108 g Tris [121.14 M]
Appendix
128
55 g boric acid [61.83 M]
7.44 g EDTA [372.24 M]
H2O ad 1000 ml
pH 8.0
D.10. Urea/TBE solution (4%)
425 g urea [60.06M]
300 ml H2O
100 ml TBE-buffer (10x)
Solubilise in a water bath at 65°C
H2O ad 900 ml
D.10. Urea/TBE solution (6%)
425 g urea [60.06M]
250 ml H2O
100 ml TBE-buffer (10x)
Solubilise in a water bath at 65°C
H2O ad 850 ml
E. Chemicals
Agarose (Invitrogen, Paisley, USA)
Ammonium persulfate (APS) ≥ 99.8% (Sigma-Aldrich Chemie GmbH,
Taufkirchen, Germany)
Borid acid ≥ 99.8%, p.a. (Carl Roth GmbH & Co, Karlsruhe, Germany)
Bromophenole blue (Merck KgaA, Darmstadt, Germany)
DMSO ≥ 99.5%, p.a. (Carl Roth GmbH & Co, Karlsruhe, Germany)
dNTP-Mix (Qbiogene GmbH, Heidelberg, Germany)
dNTP-Mix (Roche Diagnostics GmbH, Heidelberg)
EDTA ≥ 99%, p.a. (Carl Roth GmbH & Co, Karlsruhe, Germany)
Ethidium bromide (Carl Roth GmbH & Co, Karlsruhe, Germany)
Appendix
129
Ethyl alcohol (AppliChem, Darmstadt, Germany)
Formamide ≥ 99.5%, p.a. (Carl Roth GmbH & Co, Karlsruhe, Germany)
Natriumdihydrogenphosphat (Biochrom AG, Berlin, Germany)
Paraffin (Merck KgaA, Darmstadt, Germany)
PCRX Enhancer System (Invitrogen GmbH, Karlsruhe, Germany)
Rotiphorese®Gel40 (Carl Roth GmbH & Co, Karlsruhe, Germany)
SephadexTM G-50 Superfine (Amersham Biosciences, Freiburg, Germany)
TEMED 99%, p.a. (Carl Roth GmbH & Co, Karlsruhe, Germany)
Tris PUFFERAN® ≥ 99.9%, p.a. (Carl Roth GmbH & Co, Karlsruhe, Germany)
Urea ≥ 99.5%, p.a. (Carl Roth GmbH & Co, Karlsruhe, Germany)
F. Enzymes
Taq-DNA-Polymerase 5 U/µl (Qbiogene GmbH, Heidelberg, Germany)
Taq-DNA-Polymerase 5 U/µl (Roche Diagnostics GmbH, Mannheim, Germany)
KappG-Robust-DNA-Polymerase (PeqLab, Erlangen, Germany)
G. Consumables
Thermo-fast 96 well plate, skirted (ABgene, Hamburg, Gemany)
PCR-Plate PP, nature, 96 x 0.2 ml, skirted, RNase-, DNA- and pyrogenfree (nerbe
plus, Winsen/Luhe, Germany)
Adhesive PCR film (nerbe plus, Winsen/Luhe, Germany)
Adhesive PCR film (ABgene, Hamburg, Germany)
Combitips® plus (Eppendorf AG, Hamburg, Germany)
Pipette tips 0.1 – 10 µl (K138.1), 0.1 – 10 µl (A407.1), 5 – 200 µl (7058.1) (Carl Roth
GmbH & Co, Karlsruhe, Germany)
Pipette tips 0.1 – 10 µl (7600) (Matrix Technologies Corporation, Lowell, USA)
Reaction tubes 1.5 ml and 2.0 ml (nerbe plus GmbH, Winsen/Luhe, Germany)
Reaction tubes 10 and 50 ml (Falcon) (Renner, Darmstadt, Germany)
Appendix
130
H. Software
BLASTN, trace archive http://www.ncbi.nlm.nih.gov
EBI toolbox http://www.ebi.ac.uk/Tools/sequence.html
MERLIN version 1.1.2 http://www.sph.umich.edu/csg/abecasis/Merlin
Order of primers MWG Biotech-AG, Ebersberg (https://ecom.
mwgdna.com/register/index.tcl
Order of enzymes http://www.neb.com/nebecomm/products/
categories.asp
Primer design http://frodo.wi.mit.edu/cgi-bin/primer3/primer3
_www.cgi
Repeat Masker http://www.repeatmasker.genome.washington.edu/
Sequencher 4.7 GeneCodes, Ann Arbor, MI, USA
Spidey http://www.ncbi.nlm.nih.gov/IEB/Reasearch/Ostell/
Spidey/index.html
SUN Ultra Enterprises 450 Sun Microsystems
SUN FIRE V490 Sun microsystems
CHAPTER 10
List of publications
List of publications
132
10 List of publications
Menzel J, Distl O (2010). Unusual vascular ring anomaly associated with a persistent
right aortic arch and an aberrant left subclavian artery in German Pinschers. The
Veterinary Journal, accepted for publication.
Menzel J, Distl O (2010). Prevalence and formation of primary cataracts in the
German Pinscher population in Germany. Veterinary Ophthalmology, accepted for
publication.
Menzel J, Philipp U, Distl O: Molekulargenetische Untersuchung des persistierenden
rechten Aortenbogens beim Deutschen Pinscher. DGfZ/GfT conference in Gießen,
Germany, 16.-17.09.2009
CHAPTER 11
Acknowledgements
Acknowledgements
134
11 Acknowledgements
First of all I wish to thank Prof. Dr. Dr. Ottmar Distl, the supervisor of my thesis, for
offering me the opportunity to work on an interesting dissertation and for his
academic guidance and support in the course of this work.
I wish to express my appreciation to the Pinscher-Schnauzer-Klub 1895 e.V. for
providing the blood samples, the eye examination sheets and the pedigree data.
Especially I want to thank Mrs. Koch, Mrs. Paech, Mrs. Baumann and Mr. and Mrs.
de Lange for their engagement for this work and for their help with getting the blood
samples and the pedigree data.
I want to thank the veterinary ophthalmologists of the Dortmunder Kreis for providing
the current examination data.
I am also very grateful to Dr. habil. Ute Philipp and Dr. Anne Wöhlke for their always
open ears for my questions.
I am very grateful to Heike Klippert-Hasberg and Stefan Neander for teaching me the
laboratory techniques and for their support during the work in the laboratory.
I want to thank Jörn Wrede for his help with computer problems and with the
statistical analyses, Ingomar Schwan for the graphical assistance and C. Mrusek and
D. Böhm for their support concerning administrative questions.
My special thanks go to all colleagues and friends of the Institute for Animal Breeding
and Genetics of the University of Veterinary Medicine Hannover for their support,
humour and the friendly atmosphere.
Very special thanks go to my family and my friends. Thank you for your support in the
last year.
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