Molecular genetic analysis of bilateral convergent strabismus with exophthalmus in German Brown cattle

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Molecular genetic analysis of bilateral convergent strabismus with exophthalmus in

German Brown cattle

INAUGURAL-DISSERTATION zur Erlangung des Grades einer DOKTORIN DER VETERINÄRMEDIZIN

(Dr. med. vet.)

durch die Tierärztliche Hochschule Hannover

Vorgelegt von

Stefanie Hedwig Mömke aus Quakenbrück

Hannover 2004

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Scientific supervisor: Univ.-Prof. Dr. Dr. O. Distl

Examiner: Univ.-Prof. Dr. Dr. O. Distl Co-examiner: Univ.-Prof. Dr. H.-J. Hedrich

Oral examination: 19.11.2004

This work was supported by grants from the German Research Council, DFG, Bonn, Germany (DI 333/7-2).

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Dedicated to my family

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Parts of this work have been submitted for publication in the following journals:

1. Animal Genetics

2. Cytogenetic and Genome Research 3. The Veterinary Journal

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1 Introduction 2

2 Bilateral strabismus with exophthalmus (BCSE) in cattle: a molecular

genetic approach 5

– 2.1 Introduction 5

– 2.2.1 Anatomy of structures providing eye-movement 5

– 2.2.2 Strabismus and exophthalmus in cattle 6

– 2.2.3 Clinical signs of bilateral convergent strabismus with

exophthalmus (BCSE) in cattle 7

– 2.2.4 Differential diagnoses for BCSE 8

– 2.2.5 Occurrence and prevalence of bovine strabismus 9

– 2.2.6 US Brown Swiss and milk production trait associations 10

– 2.2.7 Mode of inheritance 11

– 2.2.8 Histolopathological findings 12

– 2.2.9 Human paralytic strabismus in comparison to bovine BCSE 13

– 2.2.10 Whole genome scans as alternative approaches 15

2.2.11 Comparative genomics 16

– 2.3 Conclusions 17

3 Genome-wide search for markers associated with BCSE in German

Brown cattle 19

– 3.2. Material and methods 20

– 3.2.1 Sampling and pedigree structure 20

– 3.2.2 Marker selection and three step analysis 22

– 3.2.3 Linkage analysis 23

– 3.3 Results 23

– 3.3.1 Quality of the used marker set 23

– 3.3.2 Whole genome scan 26

– 3.3.3 Mapping of putative BCSE loci with flanking markers 26

– 3.4 Discussion 27

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4 Physical mapping of the KCNJ8, MRPS35 and PRPH genes on bovine chromosome 5 by fluorescence in situ hybridisation and confirmation

by RH mapping 35

– 4.2 Material and methods 35

– 4.2.1 Identification of BAC clones containing the genes 35

– 4.2.2 Fluorescence in situ hybridisation 36

– 4.2.3 Radiation hybrid (RH) mapping 36

– 4.3 Assignment of the KCNJ8 gene to bovine chromosome 5q3.2-q3.4 37

– 4.3.1 Description 37

– 4.3.2 Isolation and characterisation of the bovine KCNJ8 clone 37

– 4.3.4 Radiation hybrid mapping 38

– 4.3.5 Comment 38

– 4.4 Assignment of the PRPH gene to bovine chromosome 5q1.4 39

– 4.4.2 Isolation and characterisation of the bovine PRPH clone 40

– 4.4.5 Comment 41

– 4.5 Assignment of the MRPS35 gene to bovine chromosome 5q3.1-q3.2 41

– 4.5.2 Isolation and characterisation of the bovine MRPS35 clone 42

– 4.5.5 Comment 43

5 A comparative radiation hybrid map of bovine chromosome 5q1.3-q2.5

with human chromosome 12q 45

– 5.2.1 Selection of genes and primer design 46

– 5.2.3 Statistical analysis 47

– 5.3 Results 47

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6 A comparative radiation hybrid map of the telomeric region of bovine

chromosome 18 to human chromosome 19q13 55

– 6.2.1 Selection of genes and primer design 55

– 6.2.2 Isolation of two BAC clones by radioactive hybridisation 57

– 6.2.3 Chromosomal location 57

– 6.2.4 Radiation hybrid (RH) mapping 58

– 6.2.5 Statistical analysis 59

– 6.3 Results 58

7 Fine mapping of two gene loci on bovine chromosomes 5 and 18 responsible for bilateral convergent strabismus with exophthalmus in

German Brown cattle 64

– 7.2.1 Pedigree material 64

– 7.2.2 Search for microsatellite markers in published BAC-end

sequences 64

– 7.2.3 Development of microsatellites from bovine BAC clones 65

– 7.2.4 Microsatellite marker analysis 67

– 7.2.5 Development of single nucleotide polymorphisms (SNPs) 68

– 7.2.6 SNP marker analysis 68

– 7.2.7 Linkage analysis 70

– 7.3 Results 70

8 Summary 82

9 Erweiterte Zusammenfassung 85

– 9.1 Einleitung 85

– 9.2 Genomscan und nicht-parametrische Kopplungsanalyse anhand von

Mikrosatelliten 86

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– 9.2.1 Material und Methoden 86

– 9.2.2 Ergebnisse 88

– 9.2.3 Diskussion 88

– 9.3 Physikalische Kartierungen mittels Fluoreszenz in situ

Hybridisierung (FISH) und Radiation Hybrid (RH)-Kartierung 89

– 9.4 Entwicklung von Mikrosatelliten und SNP Markern mit

anschließender Feinkartierung 92

10 References 96

11 Appendix 108

12 Acknowledgements 134

13 List of publications 137

– 13.1 Journal articles 137

– 13.2 Oral presentations 138

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List of abbreviations

A adenine Acc. no. accession number

ad autosomal dominant

AI artificial insemination

APS ammonium persulphate

ar autosomal recessive

AT annealing temperature

ATX amoxicillin, tetracyclin, X-Gal BAC bacterial artificial chromosome

BCSE bilateral convergent strabismus with exophthalmus BLAST basic local alignment search tool

BTA chromosome of Bos taurus

bp base pair

C cytosine

cDNA complementary desoxyribonucleic acid cM centiMorgan

cR centiRay

DAPI 4',6-diaminidino-2-phenylindole

DFG Deutsche Forschungsgemeinschaft (German Research Council) DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

dNTPs deoxy nucleoside 5’triphosphates (N is A,C,G or T) DUS divergent unilateral strabismus

E. coli Escherichia coli

EDTA ethylenediamine tetraacetic acid

EMBL European Molecular Biology Laboratory EST expressed sequence tag

F forward

FISH fluorescence in situ hybridisation G guanine

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gss genomic survey sequence HET heterozygosity HSA chromosome of Homo sapiens IBD identical by descent

IMAGE integrated molecular analysis of genomes and their expression

IRD infrared dye

ISCNDB international system for chromosome nomenclature of domestic bovids kb kilobase

LB Luria Bertani

LOD logarithm of the odds M molar

MARC U.S. Meat Animal Research Center Mb megabase

MERLIN multipoint engine for rapid likelihood inference MMU chromosome of Mus musculus

mRNA messenger ribonucleic acid MS microsatellite

mtDNA mitochondrial desoxyribonucleic acid

NCBI National Center for Biotechnology Information

NPL nonparametric linkage

OMIA online mendelian inheritance in animals OMIM online mendelian inheritance in man

p error probability

PCR polymerase chain reaction

PEO progressive external ophthalmoplegia PIC polymorphism information content QTL quantitative trait locus

R reverse

RH radiation hybrid

RPCI Roswell Park Cancer Institute rpm rounds per minute

RZPD Resource Center/Primary Database, Berlin SAS statistical analysis system

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SNP single nucleotide polymorphism

STS sequence-tagged site

T thymine

TBE tris-borate-ethylenediamine tetraacetic acid TE tris- ethylenediamine tetraacetic acid

TEMED N,N,N’,N’-tetramethylenediamine

USDA United States Department of Agriculture UV ultraviolet

wgs whole genome shotgun w/v weight to volume

X-Gal 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside

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Chapter 1 Introduction

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Introduction

Bilateral convergent strabismus with exophthalmus (BCSE) is a heritable eye defect, which is prevalent in many cattle breeds and known worldwide. German Brown cattle shows a high incidence for BCSE. The defect is characterised by a bilateral symmetric anterior-medial rotation of the eye associated with a slight to severe protrusion of the eyeball. BCSE shows a progressive course and ends up in complete blindness. Breeding with animals, which are known or suspected to be carriers of BCSE is forbidden by paragraph 11b of the German animal welfare laws, due to the severly limited use of the eyes in affected individuals. The onset of the defect can sometimes be late in life and often first signs of the defect are not noticed prior to first breeding. Thus, prevention of BCSE cannot be achieved alone by exclusion of affected animals from breeding. Consequently, a molecular genetic diagnosis of carriers is urgently needed. The objective of the present study is to identify the genomic regions harbouring the gene loci responsible for BCSE. In order to achieve this goal, a whole genome scan was performed and after that, for the genomic regions significantly linked to BCSE, comparative human-bovine maps with high resolution were constructed. Using these maps, new microsatellites and single nucleotide polymorphisms (SNPs) were developed for fine mapping the identified BCSE regions in cattle.

Overview over the chapter contents

Chapter 2 reviews the literature for BCSE in German Brown cattle and other cattle breeds, including the clinical signs, prevalence, phenotypic associations, inheritance patterns, and histopathology. Moreover, parallels to human strabismus are shown, and the advantages of comparative genomics and different molecular genetic approaches are described.

Chapter 3 describes the whole genome scan performed on ten German Brown cattle families and the linkage analysis to determine the genomic regions responsible for BCSE in German Brown cattle.

In Chapter 4 the isolation and mapping of three potential candidate genes for BCSE is described.

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Chapter 5 and Chapter 6 contain the construction of two high resolution human bovine comparative maps for two genomic regions determined by the whole genome scan (Chapter 3).

Chapter 7 shows the development of new microsatellite and SNP markers and the results of fine mapping the two genomic regions linked with BCSE.

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Chapter 2 Bilateral strabismus with exophthalmus (BCSE)

in cattle: a molecular genetic approach

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Bilateral strabismus with exophthalmus (BCSE) in cattle: a molecular genetic approach

2.1 Introduction

Bilateral convergent strabismus with exophthalmus (BCSE) is an eye disorder affecting many cattle breeds worldwide. The defect is heritable and of relatively high incidence, particularly in Holstein and German Brown cattle. BCSE can become a significant problem because of its progressive course, which leads to complete blindness. This can cause changes in the behaviour of the affected animals such as aggressiveness, shying and panic in everyday situations, or reluctance to walk to the milking parlour or to pasture. Cattle showing the clinical signs of BCSE may be suspected of being affected with bovine spongiform encephalopathy (BSE) due to similar symptoms such as insecure gait, trembling when forced to walk and shyness, as well as strabismus of the eyes. Due to the impact of BCSE on the proper use of organs, breeding with animals suspected to be carriers is not allowed by German animal welfare laws; however, sufficiently early detection is not yet possible and efficient preventive measures have to be developed to reduce the occurrence of BCSE. The objective of this chapter was to provide an overview of the phenotypic forms of strabismus in cattle, to review its prevalences, associations with other characteristics, and reported mode of inheritance, and to discuss histological findings in connection with human molecular genetic candidate genes useful for further research work.

2.2.1 Anatomy of structures providing eye-movement

While primates have frontally oriented eyeholes, in cattle the physiological angle of the axis of the eyes is 104°. This provides a field of vision of nearly 360°. Seven extraocular skeletal muscles move the eyeball: one retractor bulbi, four rectus and two oblique muscles. The interaction of these muscles adjust the eyes to particular lines of sight. The retractor bulbi muscle originates at the edge of the optic foramen, incloses the optic nerve, and attaches to the back of the eyeball. Its function is to protect the eye by retraction. The four slender rectus muscles (superior, medial,

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inferior and lateral) originate dorsal, medial, ventral and lateral of the optic foramen, respectively, and run towards the eyeball. Their tendons insert at the sclera, near the cornea. These muscles move the eyeball in every direction. The oblique muscles turn the bulbus around the axis of the eye. The inferior oblique muscle emerges from the lacrimal bone and circles ventrally around the bulbus to the temporal side of the eye.

The superior oblique muscle originates near the ethmoid foramen and runs to the nasal corner of the eye. It loops orthogonally around the trochlea and proceeds around the bulbus to the temporal side.

The motoric innervation of the eye muscles is accomplished by the oculomotor (III), trochlear (IV) and abducens (VI) nerves. The oculomotor nerve is the third cranial nerve. It enters the orbit and innervates the superior rectus, medial rectus, inferior rectus, inferior oblique muscle and the medial portion of the retractor bulbi muscles.

The trochlear nerve is the fourth and weakest cranial nerve. It innervates the superior oblique muscle. The sixth cranial nerve, the abducens, innervates the lateral rectus muscle and the lateral portion of the retractor bulbi muscle.

2.2.2 Strabismus and exophthalmus in cattle

Strabismus is defined as the permanent or temporary deviation of the eyes from their normal visual axis. The signs of strabismus can manifest congenitally or later in life.

Paralytic and non-paralytic forms of strabismus are distinguished in human and veterinary medicine.

Concomitant (non-paralytic) strabismus is due to a functional disturbance of the ocular apparatus and can be congenital or caused by infectious diseases. The angle of misalignment of the visual axes does not vary with ocular movements, and the function of individual eye muscles is usually intact.

The paralytic form of strabismus (incomitant strabismus) results from paralysis of one or more ocular muscles and leads to limited eye motion and thus to different angles of the axes of the eyes.

The most frequently observed manifestation of strabismus is convergent strabismus (esotropia), which is characterised by squinting eyes that deviate inwards, toward the nasal angle. In divergent strabismus (exotropia), the affected eye deviates outwards from its visual axis, toward the temporal angle. In other forms of strabismus the visual axis deviates upwards, downwards or obliquely. Strabismus can have many causes:

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congenital defects (e.g. aplasia of the eye-muscle nuclei), space-occupying processes within the orbit (e.g. neoplasias, haemorrhages, inflammations), neurologic diseases (e.g. meningitis, encephalitis, intracranial tumours, ischaemia, neuritis), muscular impairment (e.g. myositis), metabolic diseases (e.g. tetany, hypocalcaemic partiturient paresis, the nervous form of acetonaemia) or intoxication (e.g. phosphoric acid ester, seeds of Aesculus octandra [Magnusson et al. 1983]).

Exophthalmus is the abnormal prominence of an eyeball of normal size. It can occur in one or both eyes. In advanced stages of this defect, the cornea often desiccates.

Exophthalmus can be caused by paralysis of the muscles of the eye (e.g. a defect of the abducens nerve, lesions of the musculus retractor bulbi), space-occupying processes within the orbit (e.g. neoplasia, haemorrage, inflammation, abscess), congenital deformity of the scull or by defects of the suspension apparatus of the eyeball.

2.2.3 Clinical signs of bilateral convergent strabismus with exophthalmus (BCSE) in cattle

The signs of bilateral convergent strabismus with exophthalmus (BCSE) were first described in cattle by Koch (1875) at the end of 19^th century. BCSE is characterised by a bilateral symmetric rotation of the eyeballs in an anterior-medial direction, which results in a permanent deviation of the visual axes. Bilateral convergent strabismus is accompanied by slight to severe laterodorsal exophthalmus. According to Power (1987) this is due to the oval shape of the bovine eye, whose transverse diameter is greater than its axial diameter (Sisson 1953). Thus, the eyeball protrudes when it is rotated. Epiphora is often seen, particularly in cattle with advanced BCSE (Vogt 2000). In many cases, the visible sclera shows a dark pigmentation (Veenendal 1958; Schütz-Hänke et al. 1979). Parts of the lateral rectus muscle (Barrier and Brissot 1885) or even the retrobulbar fat pad (Schütz-Hänke et al. 1979) can become visible in severely affected animals. These defects are chronic and incurable.

The degree of deviation of both eyes from the regular visual axis can be determined by the amount of sclera permanently visible in the temporal corner of the eye. Vogt and Distl (2002) proposed a four-stage scale for classifing affected animals: stage 1, with less than 25% of the eye filled with sclera; stage 2 from 25% to 50%; stage 3 from 50% to 75%; and stage 4, with more than 75% filled (Chapter 11, Figures 1 - 4).

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Mild forms of BCSE (stage 1) are more difficult to diagnose than advanced stages.

For diagnosis of stage 1 BCSE, the animal has to be carefully watched from a distance of one to two meters for at least several minutes. The animals' sense of orientation may be intact in mildly affected individuals in spite of the limited field of vision (Miles 1932), but animals showing stage 3 or 4 of BCSE are generally disoriented (Koch 1875; Distl et al. 1991), have an insecure gait, sometimes walk in circles or even lose their balance and fall down (Jakob 1920). Handling of these animals is difficult due to their limited vision. Farmers describe affected cows as shy, leery, jumpy and wild. Behavioural changes mainly attract attention when the animals jump and kick unexpectedly upon hearing noise from behind (Schütz-Hänke et al.

1979). Affected animals have to be handled very carefully, particularly in cubicle housing and on pasture, because of their cautious movements (Distl and Gerst 2000). BCSE causes economic losses because of the animals' decreased market value and the fact that they and their progeny cannot be used as breeding animals (Distl and Gerst 2000).

2.2.4 Differential diagnoses for BCSE

While BCSE is clearly a genetic defect, several similar clinical pictures caused by other factors have been described in the literature. Zschokke (1885) carried out a pathological examination in a cow with manifest strabismus and the eyeballs rotated medially 90° from the anatomical visual axis. He detected a bilateral angioma at the foramen orbitorotundum within the orbit. The pressure of this tumour caused paralysis of the abducens nerve. An extreme exophthalmus combined with convergent strabismus was observed by Röder (1890) and Göring (1898). They found these symptoms to be connected with Morbus Basedow (Graves' Disease). In those cases the squinting animals showed typical symptoms of hyperthyroidism (e.g.

struma, tachycardia and dilatation of the heart). Dexler (1891) observed bilateral convergent strabismus with exophthalmus in four animals of different breeds. He attributed the symptoms to a hypertrophic retrobulbar corpus adiposum which led to partial or complete paralysis of the lateral rectus muscle. Magnusson et al. (1983) reported the case of a calf which developed bilateral dorsomedial strabismus after being fed with seed of Aesculus octandra Marsh. Ventromedial strabismus was found in four animals showing clinical coccidiosis (Jubb 1988).

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Bovine leukosis occasionally causes tumours which sometimes affect the central nervous system and lead to strabismus with exophthalmus, if their position is retrobulbar (Power 1987). Furthermore, Möller (1910) and Jakob (1920) described different tumours, orbital bone defects, traumatic injuries and a small orbit which caused strabismus and exophthalmus. Distl and Scheider (1994) reported a full sib pair of male Highland cattle showing divergent unilateral strabismus (DUS), which is assumed to be inheritable. The reason for this eye defect was a 40° ventral displacement of the insertion of the lateral rectus muscle. Julian (1975) described a case of divergent bilateral strabismus with hydrocephalus in a Holstein calf (1975). At birth the calf showed strabismus along with several other abnormalities. When the calf was euthanised at two months of age, the eyes returned to their normal position.

2.2.5 Occurrence and prevalence of bovine strabismus

BCSE has been observed in different cattle breeds, including German Brown, Jersey, Shorthorn, Ayrshire, Bulgarian Grey, Irish Holstein Friesian, German Fleckvieh, German Black and White, and Dutch Black Pied (Distl et al. 1991; Distl and Gerst 2000; Holmes and Young 1957; Mintschev 1965; Power 1987; Regan et al. 1944;

Vogt and Distl 2002). Generally, no signs of the defect are present at birth, but develop later in life. According to Holmes and Young (1957), the earliest manifestation of the defect is usually at the age when the heifers are in calf and often not until after calving, although those investigators also report one calf affected at birth. Regan et al. (1944) observed BCSE earliest in one six-month-old calf, but all other animals were at least one year old. First symptoms of BCSE were found in cattle at least one year old by Gerst and Distl (1997 and 1998), who also found it impossible to ascertain an age limit after which all affected animals would have started showing symptoms of BCSE. The condition generally shows a progressive course that advances at an individual speed and which may be interrupted by long, apparently stable periods (Holmes and Young 1957). Given a sufficently long lifetime, affected animals can sooner or later become completely blind.

The incidence of BCSE in German Brown cattle was estimated by Gerst and Distl (1997) to be 0.9 % in adult cows and 0.1% in young animals. Due to the smaller corpus of data for the breeds German Black and White and German Fleckvieh, only tendencies for incidences were estimated for these breeds.

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However, the incidence BCSE in German Black and White cattle seemed to be higher and that of Fleckvieh lower than in German Brown cattle. Vogt and Distl (2002) analysed the influence of an unproven German Brown sire affected by BCSE on his offspring and proved that there was a significant relationship between the paternity of this sire and the occurrence of BCSE in his progeny. The incidence of BCSE was 8.33% in the decendants of this sire, which was used for artificial insemination (AI) in German Brown dairy cattle. This incidence is much lower than the expected 50% for an autosomal dominant inheritance and a heterozygote carrier, and it was assumed that further genes influence the occurrence or age of manifestation of BCSE (Vogt 2000).

2.2.6 US Brown Swiss and milk production trait associations

It is remarkable that the proportion of US Brown Swiss blood in affected German Brown animals was up to 7% higher than in unaffected animals of this breed (Gerst and Distl 1998), and a potential association was suspected between BCSE and the incrossings of US Brown Swiss bulls. However, in a subsequent study, Vogt and Distl (2002) did not find a significant influence of the percentage of US Brown Swiss blood on the occurrence of BCSE in data from about 130 herds. Therefore it is not clear whether the spread of BCSE is caused by the intensive use of US AI bulls in the German Brown population. The defect definitely cannot be caused solely by incrossing of US Brown Swiss sires, since BCSE was also observed in maternal families without US Brown Swiss blood (Distl 1993).

No associations were found in German Brown cattle between BCSE and the milk performance traits milk, fat and protein yield or content. Nor was the prevalence of BCSE in cows associated with higher or lower breeding values for milk production traits, so it is unlikely that there was a selection advantage for cows with BCSE (Distl and Gerst 2000; Vogt 2000). Therefore it seems rather improbable that there was an indirect selection for BCSE caused by higher milk performance in affected cows. It appears unlikely that there is a close genetic linkage of the defect allele for BCSE and a gene for milk yield. Thus, bovine chromosomes other than 1, 6, 9, 10 and 20 which have already been mapped for quantitative trait loci for milk production traits, may be assumed as the most probable candidates for containing the BCSE gene or genes (Distl and Gerst 2000).

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2.2.7 Mode of inheritance

As early as 1885, BCSE in cattle was assumed to be an inherited defect. Barrier and Brissot (1885) described the case of a cow with one decendant showing a similar occurrence of strabismus and exophthalmus. Jakob (1920) advised farmers to exclude animals affected with BCSE from breeding. However, both the mode of inheritance as well as the number of contributing genes was controversially discussed for several decades. Regan et al. (1944) were the first to collect systematic records on this defect. They compared the ancestry of two male and seven female affected animals of the Jersey cattle herd owned by the California Agricultural Experiment Station. Most of the affected animals were inbred (sire- daughter matings) progeny of three different, apparently unaffected sires. The progeny of bulls from strabismus-free lines mated with affected cows did not show the defect. Thus, Regan et al. (1944) supposed that strabismus in cattle was caused by one autosomal recessive gene. This thesis was only partly affirmed by Holmes and Young (1957), who observed BCSE in three groups of Shorthorn and Ayrshire cattle, which included more than 20 affected individuals in all. Those investigators could not exclude the possibility of a recessive gene causing BCSE because their material was not sufficiently extensive. Using regressive logistic models of segregation analysis, Distl et al. (1991) examined 107 animals of the German Brown cattle breed and postulated a major gene model influenced by additively acting genes, taking into consideration environmental and polygenic effects. Complex segregation analysis was employed to study additional 10 pedigrees, including 184 German Brown individuals (Distl 1993). The results showed that an autosomal dominant major gene was the most likely explanation for the segregation of BCSE- affected cattle within the pedigrees when ascertainment was corrected. Gerst and Distl (1997) proposed that the defective allele segregated mainly within cow families and herds. The segregation analysis performed by Gerst (1996) showed a single autosomal dominant mode of inheritance with an incomplete penetrance of 70%. For this model the frequency of the BCSE gene was estimated as f = 0.008 for the available cattle population. Vogt and Distl (2002) supposed that complete penetrance for a single autosomal dominant gene causing the disease is unlikely, due to the variable age of manifestation of BCSE in cattle.

A mitochondrial DNA defect responsible for BCSE might be an alternative hypothesis

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for the mode of inheritance of BCSE. This latter hypothesis can be excluded only by showing transmission of BCSE from an affected bull to his progeny. Our unpublished data including two German Brown AI sires affected by BCSE with more than 40 affected daughters out of about 35 different herds did not provide evidence for maternal cytoplasmatic inheritance. Nearly 50% of the examined 75 descendants of these two sires were affected by BCSE.

2.2.8 Histolopathological findings

Knowing the pathogenesis of a defect can help to assign it to a specific gene that causes the same or similar findings in other species. Therefore histopathological examinations can be most helpful. In the case of BCSE-affected animals, the defect was suspected in the lateral rectus muscles (Barrier and Brissot, 1885) or in the supplying nerves and appropriate nuclear regions (Jakob 1920). Mintschev (1965) diagnosed BCSE in Bulgarian Grey cattle and came to the conclusion that the defect was probably caused by infranuclear lesions of the abducens nerves.

Pathomorphological investigation by Schütz-Hänke et al. (1979) revealed no differences in the eyes, eye muscles and the N. abducens between affected and unaffected individuals. However, those investigators' histomorphological examinations of the nucleus of the abducens nerve showed that the number of nerve cells in both nuclear regions of this nerve is decreased in animals with symptoms of BCSE and that this induces paresis of the lateral rectus muscles and the lateral part of the retractor bulbi muscles. Histological examination on the lateral and medial rectus muscles of affected cattle eyes revealed “ragged red fibres”, which are indicators for muscle defects and can be associated with mitochondrial DNA defects (Vogt 2000). Since "ragged red fibres" are not exclusively signs of mitochondrial DNA defects but can also be shown in other defects in the respiratory chain of the muscle, clarification of the pathogenesis depends on molecular genetic approaches and/or examination of tissue sections by electron microscopy for detection of deformed organelles, characteristically arranged cristae and para-cristalline inclusions.

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2.2.9 Human paralytic strabismus in comparison to bovine BCSE

It has become apparent that there are extensive genetic homologies between the human and even distantly related species. Great progress has been made in the comparative gene map between humans and cattle, so that syntenic genomic regions can be identified with high precision. Genes causing defects, e.g. strabismus, that have already been identified in man can be used as candidate genes for the clarification of BCSE in cattle.

Human congenital-infantile esotropia is not connatal, but develops in the first few weeks or months after birth (Nixon et al. 1985). Examination of 39,227 children in Bethesda, MD, USA, from gestation to the age of seven years showed that esotropia developed in 3.0% of them (Chew et al. 1984). Progressive external ophthalmoplegia (PEO) in man shows striking similarities to BCSE in cattle. PEO refers to a group of disorders characterised by ptosis and slowly progressive bilateral immobility of the eyes (Sorkin et al. 1997), and is considered to be the most frequent form of mitochondrial encephalomyopathies in man (Deschauer et al. 2001). In many cases the onset of the disease is in adolescence or adulthood. Based on age of onset and severity of clinical symptoms, patients with PEO are divided into three groups. The most severe variant is called Kearns-Sayre syndrome and is characterised by an infantile, childhood or adolescent onset. The second is the milder, chronic PEO with an adolescent or adult onset. The third is isolated chronic PEO with an adult onset and mild symptoms. PEO are monogenetic defects caused by mutations of different genes (Table 1): DNA polymerase gamma (POLG) (van Goethem et al. 2001), solute carrier family 25A4 (SLC25A4) (Kaukonen et al. 2000; Napoli et al. 2001; Komaki et al. 2002) and chromosome 10 open reading frame (C10orf2) (Spelbrink et al. 2001).

Furthermore, a mutation in the endothelial cell growth factor/platelet-derived (ECGF1) gene causes a subform of PEO (Vissing et al. 2002). Since all proteins required for replication of the mitochondrial genome are encoded by nuclear genes, defects in these genes will cause mtDNA loss or deletion, which leads to tissue dysfunction (Suomalainen and Kaukonen, 2001). Because of their high energy consumption and dependence on oxidative energy, ocular tissues are affected especially often by mitochondrial defects (Mojon, 2001).

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Table 1 Genes responsible for progressive external ophthalmoplegia (PEO) in humans, and their genomic locations in man and cattle

Gene Function Location

human

Location cattle POLG Encodes for DNA polymerase involved in replication

of mitochondrial genome (Clayton 1982)

15q25 21q17- q22 SLC25A4 Catalysation of exchange of ADP and ATP across

mitochondrial internal membrane (Li et al. 1989)

4q35 27q14- q15 C10orf2 Involved in mammalian mitochondrial DNA

maintenance (Spelbrink et al. 2001)

10q24 26q13- q21 ECGF1 Promotion of angiogenesis in vivo and stimulation of

the in vitro growth of endothelial cells, limitation of glia cell proliferation (Hagiwara et al. 1991; Stenman et al. 1991, 1992)

22q13 -

The mode of inheritance of the various gene defects causing PEO is autosomal dominant (adPEO) or autosomal recessive (arPEO) (Table 2). Furthermore, mitochondrial point mutations have been suggested which are passed on maternally (Deschauer et al. 2001). In a sporadic case of PEO, Van Goethem et al. (2003) also identified heterozygosity for a 1031G-A transition in the C10orf2 gene, which resulted in an arg334-to-gln mutation, and heterozygosity for a gly884-to-ser mutation in the POLG gene, which indicate a digenic mode of inheritance. A survey of the mutations causing PEO in man is given in Table 2 for the genes POLG, SLC25A4, C10orf2 and ECGF1.

The three genes with dominantly acting mutations causing PEO in humans were chosen as candidate genes for BCSE in cattle by Hauke et al. (2003). After localisation of POLG, SLC25A4 and C10orf2 on bovine chromosomes BTA 21, BTA 27 and BTA 26, microsatellite markers were developed and tested for allelic cosegregation with the BCSE phenotype. Neither these markers nor evenly distributed microsatellite markers on the respective bovine chromosomes showed significant linkage with BCSE. Thus these candidate genes could be excluded as responsible for bovine BCSE.

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Table 2 Examples of mutations in the genes POLG, SLC25A4, C10orf2 and ECGF1 causing symptoms of PEO

Gene Mode of inheritance Mutation

Autosomal dominant A-to-Gtransition -> tyr955-to-cys (Van Goethem et al. 2001) G-to-A transition -> ala467-to-thr (Van Goethem et al. 2001) POLG

Autosomal recessive

T-to-G transversion -> leu304-to-arg (Van Goethem et al.

2001)

G-to-C transversion -> ala-to-pro (Kaukonen et al. 2000) G-to-A transition -> val-to-met (Kaukonen et al. 2000) T-to-C transition -> leu98-to-pro (Napoli et al. 2001) SLC25A4 Autosomal dominant

A-to-G heterozygous transition -> asp104-to-gly (Komaki et al. 2002)

Duplication at nucleotides 1053-1092, resulting in a duplication of amino acids 352-362 of twinkle (Spelbrink et al. 2001) G-to-C transversion -> ala475-to-pro (Spelbrink et al. 2001) G-to-C transversion -> arg354-to-pro (Spelbrink et al. 2001) C10orf2 Autosomal dominant

T-to-C transition -> leu381-to-pro (Spelbrink et al. 2001) A-to-C transversion -> glu289-to-ala (Nishino et al. 1999) A-to-C transversion -> gly145-to-arg (Nishino et al. 1999) ECGF1 Autosomal recessive

A-to-G transition -> lys222-to-ser (Nishino et al. 1999)

2.2.10 Whole genome scans as alternative approaches

A whole genome scan can be used to detect markers significantly linked to the BCSE phenotype. This approach is based on highly informative microsatellites evenly spread over all chromosomes with an average distance of less than 20 cM. The most recent linkage map of Kappes et al. (1997) contains 1250 markers covering 2990 cM with a mean marker distance of 2.5 cM. The updated bovine map with about 1250 microsatellites is freely available in the internet (URL: http://sol.marc.usda.gov).

However, much denser maps are needed in the second step of the linkage study to progress from the initial mapping of the disease to one or more specific bovine chromosomes to the identification of the precise chromosomal region. The average spacing of markers in such maps should be between 0.5 and 2 cM. Once a genomic region of the size below 2–3 cM has been successfully identified for BCSE, positional candidate genes from the conserved chromosomal region in man can be selected for testing cosegregation with BCSE.

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2.2.11 Comparative genomics

Candidate genes for BCSE may code for ionic channels, hormones, enzymes, metabolic factors and/or their receptors involved in the development of cranial nerves or eye muscles. As the number of potential candidate genes in the whole genome is far too large to be included in a study, very precise identification of the position of the genomic region harbouring the BCSE causing gene(s) is necessary. Then a candidate gene can be chosen from the homologous human genomic region based on the comparative human-bovine map. This positional candidate gene has to be considered to be causal for BCSE due to its function or pattern of expression. Of all mammals, human and mouse are the species whose genomes are best researched.

With the development of highly resolved human-bovine comparative maps, the identification of genomic regions containing candidate genes known in human or mouse becomes feasible for the bovine genome.

After the early large genome surveys which showed rough chromosomal homologies and breakpoints (Hayes 1995; Solinas-Toldo et al. 1995; Chowdhary et al. 1996), and the more detailed radiation hybrid (RH) maps (Williams et al. 2002; Band et al.

2000), Larkin et al. (2003) constructed a comparative human-bovine map by producing about 60,000 bovine BAC end sequences out of 40,224 cattle BAC clones.

Using BLASTN, 29.4% and 10.1% significant hits could be anchored to human and mouse genome sequences, respectively. Using these bovine BAC end sequences, Everts-van der Wind et al. (2004) were able to further refine the comparative human- bovine map by identification of 195 conserved segments with their breakpoints in the bovine map.

Once a positional candidate gene has been localised, single nucleotide polymorphisms (SNPs) can be developed for this gene and the flanking genes to be used in linkage analysis. In the case of BCSE, cosegregation of SNPs with BCSE can easily be detected when heterozygous SNPs within the positional candidate gene have been identified for the affected AI bulls. In this way it will be possible to characterise the yet unknown BCSE-causing gene(s) in cattle.

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2.3 Conclusions

Bilateral convergent strabismus with exophthalmus (BCSE) is a dominantly inherited defect in cattle which usually cannot be diagnosed in calves, heifers or young bulls, so these animals will spread the defect in the cattle population before they can be excluded from breeding. The development of a gene test is necessary to identify affected animals at an early age. Three candidate genes causing dominantly inherited progressive external ophthalmoplegia (adPEO) in man have been excluded.

A whole genome scan has to be completed for the BSCE-causing genes to be identified. Comparative genomics can then be used as a very effective approach towards unravelling the genetic basis of bovine BCSE. When the genes with their causal mutations for BCSE are identified, breeding strategies can be developed to eradicate this defect in cattle. Furthermore, new insights may be gained into the causes and pathogenesis of strabismus, possibly leading to therapeutic measures.

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Chapter 3 Genome-wide search for markers associated

with BCSE in German Brown cattle

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Genome-wide search for markers associated with BCSE in German Brown cattle

3.1 Introduction

The bilateral convergent strabismus with exophthalmus (BCSE) is a widespread inherited defect in several cattle populations. Affected cattle shows a permanent anterior-medial rotation of both eyes with a bilateral symmetric protrusion of the eyeballs. The defect usually does not manifest prior to an age of six months and sometimes not until after first calving. The condition is progressive and in advanced stages the deviation of the eyeballs from the proper optic axis is so strong, that pupils disappear in the nasal angles of the eyes, what leads to blindness. In the course of the disease four different stages can be distinguished: in the first stage the temporal angle of the eye is filled out by sclera up to 25 %, in the second and third stage up to 50 and 75 %, respectively, and in the fourth stage more than 75 % of the temporal angle of the eye is filled with sclera. Distl et al. (1991) used six pedigrees with altogether 107 animals of the breed German Brown Swiss to test for the mode of inheritance of BCSE. The complex segregation analysis showed that a major gene model with additively acting genes was the most likely explanation for the occurence of BCSE in these pedigrees. A further complex segregation analysis gave evidence for a single autosomal dominant major gene responsible for the phenotypic expression of BCSE (Distl 1993). On this account we performed a genome-wide search for BCSE associated microsatellite markers using two paternal half-sib pedigrees of affected German Brown sires and mostly affected descendants. In addition, eight German Brown families mainly consisting of affected cows were analysed. The objective of the present analysis was to identify genomic regions linked with the BCSE phenotype in German Brown cattle.

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3.2. Material and methods

3.2.1 Sampling and pedigree structure

For the linkage analysis we collected blood, semen or hair samples from 131 German Brown cattle individuals belonging to ten families segregating for BCSE (Table 1). Of these animals 72 belonged to two paternal half-sib families. The first half-sib family consisted of the affected sire, 16 affected daughters and nine dams, of which three were affected. The second half-sib family included the affected sire and 26 affected and 19 nonaffected daughters. The remaining eight families segregating for BCSE contained affected females and their relatives (Figure 1). Because of the late onset of the disease we preferentially included affected individuals in our analysis. Most animals showed a first or second stage of BCSE. Regarding all maternal pedigrees (families 1-8), seven non-affected sires occurred in more than one family.

In total, the pedigrees contained 53 male and 208 female animals and 126 founders.

The average family size was 27.3 individuals, with a maximum of 89 and a minimum of 5. In the average, the families included 3.9 generations, ranging from 2 to 8. The prevalence of BCSE was 70.2 % in the genotyped animals. The average examination age, at which a strabismus was detected was 6.1 for the maternal families (families 1-8), 6.9 for family 9 and 3.7 for family 10.

Table 1 Family sizes and prevalence of BCSE. The families 1 to 8 are maternal families segregating for BCSE, 9 and 10 are half-sib families of affected sires

Maternal families

Half-sib families

Total¹

Family 1 2 3 4 5 6 7 8 9 10

Number of animals 31 45 7 13 5 14 11 25 33 89 261 Number of animals affected by BCSE 8 11 3 5 2 4 3 9 20 28 93 Prevalence of BCSE (total %) 25.8 24.4 42.9 38.5 40.0 28.6 27.3 36.0 60.6 31.5 35.6 Genotyped individuals 15 18 6 7 2 6 5 12 26 46 131 Genotyped individuals affected by

BCSE

8 11 3 5 2 4 3 9 20 27 92

Prevalence of BCSE (genotyped, %) 53.3 61.1 50.0 71.4 100 66.7 60.0 75.0 76.9 58.7 70.2

1Seven sires are present in more than one family (Family 1 to 8). This causes total values smaller than the sum of family sizes.

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IV

III ^VII

V VI

VIII

800 813 809 800

804 40 34 118

33 119 819

809 800

78 810 806 41 37

767 303 87 31 25 477 801 802 801 787

782 27 807 48 43 55

802 79 800 26

86 115 125 51 338

821 804 818 802 808 800

353 805 116 114 106 800 71 332

56 444 824 806 755 107 64 772

785 738 731 102 65

II

Male Female Affected No sample available Pedigree had to be splitted

for statistic program

Figure 1 Family structure of eight maternal families (I-VIII) segregating for BCSE. The families are specified by Roman numerals.

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Genomic DNA from EDTA blood samples was extracted by using the QIAamp 96 Spin Blood Kit (Qiagen GmbH, Hilden, Germany). For hair specimen, the DNeasy®

Tissue Kit (50) (Qiagen GmbH, Hilden, Germany) was used and for semen samples, the Nucleon BACC2-Kit for blood and cell cultures (Amersham Biosciences, Freiburg, Germany) was applied.

3.2.2 Marker selection and three step analysis

For the whole genome scan we selected 164 highly polymorphic microsatellite markers from published bovine linkage maps to achieve a uniform coverage of all bovine autosomes. Markers with a reported low heterozygosity or an uncertain location on the bovine linkage map were not used. The 164 markers were evenly distributed over all bovine autosomes (Chapter 11, Figures 5 - 9) comprising 2808.9 cM with an average pair-wise distance of 19.9 cM. We used 5.7 markers per chromosome in the average. The marker set used for scanning the bovine autosomes is presented in Chapter 11 (Table 1). The whole genome scan included families 1 to 9.

In the second step of the genome scan, six genomic regions located on six different chromosomes with error probabilities lower than p=0.1 for LOD scores were scanned with 30 additional microsatellite markers. In the average, the respective six chromosomes were now covered by 11 markers each with a mean pair-wise distance of 9.3 cM. The same families were used as in the whole genome scan. All additional markers employed for fine mapping are given in Chapter 11 (Table 2).

In the third step, all markers showing significant linkage in step two were additionally genotyped for family 10.

All PCR reactions were carried out in 12 µl reaction mixtures containing 2 µl genomic DNA (10 ng/µl), 1.2 µl 10x PCR buffer, 0.24 µ DMSO, 0.5 µl of each primer (10 pmol/µl), 0.2 µl dNTPs (5 mM each) and 0.1 µl Taq Polymerase (5 U/µl) (Qbiogene, Heidelberg, Germany). To increase efficiency, 102 primer pairs were pooled into PCR multiplex groups of two to five markers, and the 62 remaining primer pairs were amplified separately. One primer of each pair was endlabeled with fluorescent IRD700 or IRD800. For amplification, PTC 100™ or PTC 200™ thermal cyclers (MJ Research, Watertown, MA, USA) and a general PCR program with variable

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annealing temperature (AT) were used. The reaction started with denaturing all samples at 94°C for 4 min followed by an empirically determined amount of cycles (32 to 37) comprising denaturation for 30 s at 94°C, annealing for 30 s at AT (52- 60°C) and extension for 45 s at 72°C. The PCR was completed with a final cooling at 4°C for 10 min. The multiplex groups and the separately amplified PCR products were pooled according to their size and labelling and diluted with formamide loading buffer in ratios from 1:3 to 1:50, that were determined empirically and carried out prior to electrophoresis. For the analysis of the marker genotypes, the PCR products were size-fractionated by gel electrophoresis on an automated sequencer (LI-COR 4200, Lincoln, NE, USA) using 6% polyacrylamide denaturing gels (Rotiphorese®Gel 40, Roth, Karlsruhe, Germany). Allele sizes were scored against IRD 700- and IRD 800-labeled DNA ladders used as standards on every gel. Alleles were assigned by visual examination.

3.2.3 Linkage analysis

We performed a multipoint non-parametric linkage analysis by using the MERLIN Software Package version 0.10.2 (Abecasis et al. 2002). The linkage between the BCSE phenotype and markers was estimated through the proportion of alleles shared identical by descent (IBD) by affected animals (Kong and Cox 1997;

Whittemore and Halpern 1994; Kruglyak et al. 1996). The Whittemore and Halpern NPL pairs statistics, the Zmean, p-values and the LOD scores according to Kong and Cox (1997) were employed for the chromosomewise search for allele sharing among affected family members. Prior to linkage analysis family 10 was splitted into two subfamilies, because the MERLIN program was not capable to manage its size.

3.3 Results

3.3.1 Quality of the used marker set

The markers for the whole genome scan had a mean number of 5.9 alleles in our material. Compared with the published data, we observed fewer alleles. For eight markers however, the number of alleles counted was higher than in literature. The

average polymorphism information content (PIC= 1-

∑

= n i

pi 1

2 -

∑

⁻

= 1 1 n

i

∑

+

= n

i

j pi

1 2 2

p j 2

) of this

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set was 0.56 and the mean observed heterozygosity 0.60, so our marker set was highly informative and appropriate for linkage studies (Table 2). The heterozygosity of individual markers was ranging from 2% (BM741) to 88% (BSE1MS2). The PIC values per microsatellite showed a minimum of 2% (BM741) and a maximum of 82%

(BM315). The PIC was higher than 50% in 110 markers (67.1%). Only ten markers (6.1 %) showed a PIC < 25% (Table 3). The highest average number of alleles was 8.3 for BTA 23 and the lowest one was 5.0 for BTA 7 and BTA 24 (Table 4). The mean observed heterozygosity ranged from 40.0 % (BTA 16) to 75.0 % (BTA 29) and the mean PIC value ranged from 37.0 % (BTA 16) to 69.0 % (BTA 18 and BTA 27).

The highest average marker distance was reached on BTA 27 (29.2 cM) and the smallest one was calculated for BTA 26 (14.3 cM).

Table 2 Characteristics of the 164 bovine microsatellite markers on all 29 autosomes for families 1-9

Characteristic Mean Minimum Maximum

Number of alleles (literature) 9.76 2 30 Number of alleles observed 5.91 2 13 Heterozygosity (literature) (%) 61.0 13.0 88.0 Heterozygosity (%) observed 60.0 2.0 88.0 Polymorphism information content (%) 56.0 2.0 82.0 Average distance between the markers (cM) 19.9 5.2 38.5

Table 3 Distribution of the polymorphism information content (PIC) for all markers of the genome scan for families 1 - 9 and in total. Family 10 was not used for the whole genome scan and was listed for comparison with values refering to the markers on chromosome 5 and 18

PIC¹ (%) Percentage of markers per family (1-10) and in total

1 2 3 4 5 6 7 8 9 Total² 10³

< 25 12.2 9.1 17.7 12.2 15.2 10.4 14.6 11.6 9.1 6.1 13.3 25 - 50 26.8 29.3 28.7 36.6 45.7 38.4 35.4 30.5 30.5 28.8 46.7

> 50 61.0 61.6 53.6 51.2 39.1 51.2 50.0 57.9 60.4 67.1 40.0

1Polymorphism information content (%)

2Total values for all markers of the whole genome scan respecting families 1 to 9

3Only for markers on bovine chromosome 5 and 18

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Table 4 The marker set with mean number of alleles, heterozygosity, PIC and marker distance per chromosome as well as chromosome size and number of markers

Bovine chromosome

Average allele number

Average HET¹ (literature)

Observed average HET¹

Average PIC²

Average distance (cM)

Chr.

size (cM)³

Number of markers BTA 01 6.1 58.44 67.00 63.00 17.5 135.50 9 BTA 02 5.2 69.33 53.00 47.00 22.5 120.41 6 BTA 03 7.0 54.86 55.00 49.00 17.6 125.21 8 BTA 04 5.2 56.17 57.00 51.00 20.0 101.50 6 BTA 05 6.6 63.43 58.00 58.00 20.0 122.10 7 BTA 06 5.3 58.00 51.00 47.00 17.5 125.60 8 BTA 07 5.0 54.11 54.00 47.00 15.8 134.10 9 BTA 08 6.0 63.50 70.00 63.00 22.1 116.30 6 BTA 09 6.3 69.50 72.00 68.00 20.5 108.41 6 BTA 10 5.3 60.80 62.00 62.00 19.5 101.41 6 BTA 11 5.9 66.43 59.00 55.00 18.7 123.50 7 BTA 12 5.2 62.83 44.00 41.00 19.5 105.80 6 BTA 13 7.3 62.00 63.00 61.00 26.5 87.10 4 BTA 14 7.8 65.40 69.00 65.00 14.9 85.71 6 BTA 15 4.8 59.80 58.00 48.00 18.5 93.41 6 BTA 16 4.6 60.80 40.00 37.00 21.5 93.21 5 BTA 17 6.4 61.60 68.00 62.00 23.7 98.60 5 BTA 18 7.4 69.40 74.00 69.00 21.2 84.71 5 BTA 19 5.3 58.83 66.00 61.00 19.7 99.50 6 BTA 20 5.2 61.80 49.00 49.00 18.8 75.00 5 BTA 21 6.4 61.50 71.00 64.00 20.4 87.60 5 BTA 22 4.4 60.80 62.00 54.00 20.3 81.10 5 BTA 23 8.3 63.25 61.00 64.00 19.0 67.10 4 BTA 24 5.0 67.75 61.00 56.00 17.7 62.50 4 BTA 25 6.5 61.75 54.00 51.00 20.2 64.91 4 BTA 26 6.0 61.00 70.00 66.00 14.3 72.60 5 BTA 27 6.3 68.00 72.00 69.00 29.2 64.10 3 BTA 28 5.8 61.75 54.00 50.00 17.5 52.40 4 BTA 29 6.5 62.25 75.00 67.00 21.7 65.00 4 Average 6.0 62.20 61.00 57.00 19.9 94.98 5.7

1Heterozygosity (%)

2Polymorphism information content (%)

3Chromosome size (cM), MARC/USDA (www.marc.usda.gov)

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3.3.2 Whole genome scan

In the first step of the whole genome scan, we detected six different putative genomic regions linked with the BCSE phenotype with error probabilities for LOD scores below or equal 0.1. All putative BCSE loci were located on different chromosomes.

The loci were mapped on bovine chromosomes 5, 6, 8, 16,18, and 22 (Table 5).

Table 5 Chromosomal regions linked to BCSE with an error probability of ≤0.1 for the LOD score. LOD scores and Zmeans with error probabilities (p-values) are given

BTA¹ Position (cM)² Marker LOD score pL-value³ Zmean pz-value⁴

5 34.7 OARFCB5 0.45 0.07 0.87 0.2

6 0.0 ILSTS093 0.35 0.1 0.77 0.2

8 76.7 HEL9 0.4 0.09 0.87 0.2

16 24.5 BM121 0.95 0.02 1.77 0.04

18 84.7 TGLA227 0.62 0.05 1.49 0.07

22 81.1 BM4102 0.37 0.1 0.6 0.3

1Bovine chromosome

2centi Morgan

3Error probability for the LOD score

4Error probability for the Zmean value

3.3.3 Mapping of putative BCSE loci with flanking markers

Additional 30 markers were tested in family 1 to 9 in the second step. Significant linkage between BCSE and markers on chromsome 5, 16 and 18 was confirmed.

The loci on all other chromosomes did not show significant linkage with BCSE.

In a third step all markers on chromosomes 5, 16 and 18 were additionally tested in family 10. A linkage analysis for these markers regarding all families revealed a significant linkage for markers on chromosome 5 and also a significant linkage for markers on chromosome 18 (Tables 6 and 7). The putative QTL for BCSE on chromosome 16 was not longer confirmed.

On BTA 5, the locations of the peaks for Zmeans and LOD scores differed between the families 9, 10 and 1 to 8. For family 9, the Zmean and LOD score peaked at the marker BMC1009 at 40.6 cM. In the families 1 to 8 and 10, the highest Zmean and LOD score were at BL23 at 28.6 cM. The most likely positions for the genes causing

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BCSE are located on chromosome 5 between the markers BP1 and BL37 and on chromosome 18 in the neighbourhood of the markers TGLA227 and MS936FBN at about 84.7 cM (families 1 to 8 and 10), or near to the marker BM6507 at 78.9 cM (family 9). The 95% confidence interval on BTA 5 extends from 18.8 to 50.9 cM, on BTA 18 it ranges from 73.7 to 85.0 cM. The paternal half sib family including 16 affected descendants of one affected sire (family 9) was a highly informative pedigree. In this family, 11 siblings got the same haplotype for the linked markers on chromosome 5 from their sire. Only two half sibs (222, 287) did not show that specific haplotype (Figure 2). One sibling showed a recombination between the markers RM103 and BMC1009 and of the remaining two half sibs the inherited haplotype could not be defined. Haplotype analysis of the second genomic region for BCSE on the telomeric region of chromosome 18 revealed that 12 siblings got the same paternal haplotype, one showed a recombination between the markers BMS2785 and BM6507, two had the alternative haplotype (278, 535), and of one animal (280), the inherited haplotype could not be defined (Figure 2). In total, eight daughters inherited the susceptible haplotype on both chromosomes, five daughters on one of the both chromosomes and one animal (233) had a recombinant haplotype for both chromosomes. For three individuals (604, 566, 280) the inherited haplotype could not be determined for one chromosome but on the other chromosome these individuals showed the haplotype associated with BCSE.

For family 9, the highest Zmean reached was 2.86 for 40.6 cM at BTA 5 and 3.53 for 78.9 cM at BTA 18 (Table 8 and 9). The second paternal half-sib family (family 10) included no genotyped dams for the 45 descendants of the common sire, but due to its size it is still a valuable family. The highest Zmeans reached for BTA 5 and BTA 18 were 2.94 at 28.6 cM and 0.79 at 84.7 cM, respectively (Table 8 and 9). The remaining eight families (families 1 to 8) were not as informative. Yet, they supported the results of the first two families with highest values at 40.6 cM for BTA 5 and at 84.7 and 85 cM for BTA 18 (Table 8 and 9).

3.4 Discussion

The linkage analysis based on IBD mapping showed the existence of two putative gene loci involved in the development of BCSE in cattle. These putative BCSE loci were located on the bovine chromosomes 5 and 18, respectively. The two loci were

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Table 6 Linkage analysis for families 1-10 for bovine chromosome 5, regarding Zmean, LOD score and error probabilities (p-values)

Marker Position (cM)¹

Zmean pz-value² LOD score pL-value³

max achievable 19.33 0.0 6.55 0.0

min achievable -3.5 1.0 -0.44 0.9

BMS1095 0.0 1.6 0.05 0.97 0.02

BMS6026 6.7 0.95 0.2 0.28 0.13

BMS695 9.0 1.06 0.14 0.48 0.07

BMS610 12.8 1.04 0.15 0.5 0.07

BP1 18.8 0.41 0.3 0.09 0.3

RM103 28.6 3.51 0.0002 1.72 0.002

BL23 28.6 3.53 0.0002 1.72 0.002

AGLA293 32.0 3.46 0.0003 1.71 0.002

BM1315 32.5 3.42 0.0003 1.7 0.003

OARFCB5 34.7 3.24 0.0006 1.6 0.003

ILSTS022 38.0 2.98 0.0014 1.35 0.006

BM321 38.0 3.01 0.0013 1.38 0.006

BMC1009 40.6 3.68 0.00012 1.8 0.002

BMS1898 44.1 3.17 0.0008 1.21 0.009

BL37 50.9 0.59 0.3 0.09 0.3

BL4 51.2 0.56 0.3 0.09 0.3

BMS1617 55.6 0.41 0.3 0.06 0.3

BMS1216 75.6 0.15 0.4 0.01 0.4

BM315 100.1 0.80 0.2 0.17 0.2

BMS597 120.0 0.19 0.4 0.05 0.3

1centi Morgan

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Table 7 Linkage analysis for families 1-10 for bovine chromosome 18, regarding Zmean, LOD score and error probabilities (p-values)

Marker Position (cM)¹

Zmean pz-value² LOD score pL-value³

max achievable 19.54 0.0 6.58 0.0

min achievable -3.51 1.0 -0.44 0.9

IDVGA31 0.0 -1.27 0.9 -0.16 0.8

BMS2213 26.2 -0.69 0.8 -0.09 0.7

INRA63 48.7 -0.71 0.8 -0.08 0.7

BMS2639 57.0 -0.56 0.7 -0.07 0.7

IDVGA055 70.5 -0.13 0.6 -0.01 0.6

RME001 70.5 -0.12 0.5 -0.01 0.6

BMS2785 73.7 0.53 0.3 0.15 0.2

BMS2078 77.8 1.39 0.08 0.62 0.04

BMS6507 78.9 1.53 0.06 0.68 0.04

TGLA227 84.7 2.5 0.006 1.35 0.006

MS936FBN 85.0 2.49 0.006 1.34 0.007

1centi Morgan

mapped on BTA 5 between the markers BP1 (18.8 cM) and BL37 (50.9 cM), and on the telomeric end of BTA 18 distally to the marker BM2785 (73.7 cM). Distl (1993) proved a single autosomal dominant major gene responsible for the phenotypic expression of BCSE, so it is possible that only one of the two chromosomes carries the gene causing BCSE. The second gene could influence the grade of BCSE or the age of onset. A possible indication for this theory can be found in the paternal half-sib families (families 9 and 10). While most of the animals of family ten showed an early onset of the eye defect with about three to four years, most of the progeny in family nine developed signs of BCSE not prior to an age of six years. The heterogenity of the LOD scores for BTA 18 between these two families may be caused by the different age of onset. So bovine chromosome 18 could harbour a gene suppressing the expression or delaying the onset of BCSE.

Molecular genetic analysis of bilateral convergent strabismus with exophthalmus in German Brown cattle