Tierärztliche Hochschule Hannoverartery in German pinschers 05 2.1 Abstract 06 2.2 Introduction 06...

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


3.4 Results and Discussion 30


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


4.4 Results 56

4.5 Discussion 58


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


5.4 Results and Discussion 73


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


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


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


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


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


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


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


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


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).


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


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.


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.


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.


19

Wrede, J., Schmidt, T. 2003. OPTI-MATE Version 3.81. A management programme

useful to minimize inbreeding in endangered populations. Programme Manual.


Hannover, Germany.


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


21

Fig. 1: Contrast oesophagram of puppy 2 (ventro-dorsal projection): Highly dilated

oesophagus (A) cranial to heart base.


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:


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:


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


Hannover, Foundation, Bünteweg 17p, 30559 Hannover


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


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).



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).


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


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


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


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


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.


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.


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.


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.


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.




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.







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

r of persistent right aortic arch in the G

erman P

inscher

35

49

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


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




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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




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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


X1 gene as candidate for a rare fom

r of persistent right aortic arch in the G

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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




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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




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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




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Table 4: Non-parametric test statistics Zmean and LOD Score, their error probabilities (PZ, PL), polymorphism information


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




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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




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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)




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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




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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


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


49

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



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.


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


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.


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


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


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


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.


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


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.


60


This study was supported by the Pinscher-Schnauzer-Klub e.V. (PSK) and the

Association for Diagnosis of Inherited Eye Diseases in Animals (DOK).


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.


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


18. Barnett KC. Hereditary cataract in the Welsh Springer Spaniel. Journal of Small


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.


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


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.


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


65

Figure 1: Cumulative distribution of age at diagnosis in German Pinschers affected

by primary non-congenital cataracts (n = 40)


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




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


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.


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


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,


71

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.


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


72

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).


73

4.4 Results and Discussion


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,


74

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).


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.


75

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


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.


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


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


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


Rubin LF, Flowers RD. Cataract in Golden Retrievers. Journal of the American


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


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.


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


79

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


80

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



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

Scanning 20 candidate genes for association w

ith primary cataracts in G

erman

Pinschers

81

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



erman

Pinschers

82

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



erman

Pinschers

83



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



erman

Pinschers

84


85

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


86

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:


87

Family 2:


88

Family 3:


89

Family 4:


90

Family 5:


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


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.



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.


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.




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.





General discussion

99



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


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


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


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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


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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


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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


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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


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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.


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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


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.


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-


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


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


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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


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


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


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)

Appendix

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


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


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.

Tierärztliche Hochschule Hannoverartery in German pinschers 05 2.1 Abstract 06 2.2 Introduction 06...

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