Aus dem Institut für Tierzucht und Vererbungsforschung der Tierärztlichen Hochschule Hannover ______

Physical and comparative mapping of candidate genes on the horse genome

INAUGURAL-DISSERTATION zur Erlangung des Grades einer DOKTORIN DER VETERINÄRMEDIZIN (Dr. med. vet.) durch die Tierärztliche Hochschule Hannover

vorgelegt von Dorothee Stübs geb. Müller aus Heidelberg

Hannover 2006

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

Examiner: Univ.-Prof. Dr. Dr. habil. O. Distl Co-Examiner: Prof. Dr. Dr. habil. H. Niemann

Oral examination: 23.11.2006

This work was supported by grants from the German Research Council, DFG, Bonn,

Germany (DI 333/12-1).

To my beloved husband and our son

Parts of this work have been accepted for publication or have been published in the following journals:

1. Animal Genetics 2. Cytogenetic and Genome Research 3. Archives for Animal Breeding

Table of contents

Chapter 1 Introduction 13

Chapter 2 Mapping the horse genome and its impact on equine genomics for identification of genes for monogenic and complex traits 17 Abstract 19 Zusammenfassung 20 Introduction 20 Organization of the horse genome 21 Cytogenetic map 22 Synteny map/ Radiation hybrid map 22 Comparative map 23 Linkage map 25 Chromosomal abnormalities 26 Coat colours 26 Equine genetic diseases and the development of gene tests 31 Monogenic equine diseases 31 Genetically complex equine diseases 33 Conclusions and perspectives 36 References 37

Chapter 3 Assignment of the COMP gene to equine chromosome 21q12-q14 by FISH and confirmation by RH mapping 53 Source/description 55 Primer sequences 56 Chromosomal location 56 Radiation hybrid mapping/ PCR conditions 57 Comment 57

Acknowledgements 57 References 57 Figure 58

Chapter 4 Physical mapping of the PTHR1 gene to equine chromosome 16q21.2 59 Source/description 61 Primer sequences 62 Chromosomal location 62 Radiation hybrid mapping/ PCR conditions 63 Comment 63 Acknowledgements 64 References 64 Figure 65

Chapter 5 Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5 And SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping 67 Source/description 69 BAC library screening/ sequence analysis 71 Chromosomal location 72 Radiation hybrid mapping 72 Comment 73 Acknowledgements 73 References 73 Figures 74 Table 1 77 Table 2 80

Chapter 6 Physical mapping of the ATP2A2 gene to equine chromosome 8p14->p12 by FISH and confirmation by linkage and RH mapping 83

Rationale and significance 85 Materials and methods 85 BAC isolation and characterization of the equine ATP2A2 clone 85 PCR conditions and microsatellite analysis 87 Chromosome preparation 87 Fluorescence in situ hybridization 87 Radiation hybrid (RH) mapping 88 Linkage mapping 89 Results and discussion 89 Polymorphism 89 Chromosomal location 89 Acknowledgements 90 References 90 Figure 91 Table 92

Chapter 7 Assignment of the COL8A2 gene to equine chromosome 2p15-p16 by FISH and confirmation by RH mapping 93 Source/description 95 Primer sequences 96 Chromosome location 96 Radiation hybrid mapping 97 Comment 97 Acknowledgements 98 References 98 Figure 98

Chapter 8 Assignment of the TYK2 gene to equine chromosome 7q12-q13 by FISH and confirmation by RH mapping 99 Background 101 Procedures 101

Results 103 Acknowledgements 103 References 104 Figure 104

Chapter 9 Assignment of CART1, COL9A3, GNRH2 and TCIRG1 to equine chromosomes by FISH and confirmation by RH mapping 105 Background 107 Procedures 109 Results 110 Acknowledgements 112 References 112 Figures 115 Tables 117

Chapter 10 General discussion 119

Chapter 11 Summary 127

Chapter 12 Zusammenfassung 131

Appendix 139

List of publications 149

Acknowledgements 152

List of abbreviations

A adenine BAC bacterial artificial colony BLAST basic local alignment search tool BLASTN basic local alignment search tool nucleotide bp base pairs C cytosine cDNA complementary desoxyribonucleic acid cen centromer CHORI Children`s Hospital Oakland Research Institute 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) ECA Equus caballus autosome EDM multiple epiphyseal dysplasia EMBL European Molecular Biology Laboratory F forward FISH fluorescence in situ hybridisation G guanine GTG giemsa trypsin giemsa HSA Homo sapiens autosome IMAGE integrated molecular analysis of genomes and their expression INRA Institut National de la Recherche Agronomique ISCNH international system for chromosome nomenclature of the domestic horse kb kilobase LOD logarithm of the odds

Mb megabase MED multiple epiphyseal dysplasia mRNA messenger ribonucleic acid MS microsatellite

NaH2PO4 Natriumdihydrogenphosphat

Na2HPO4 Dinatriumhydrogenphosphat NCBI National Center for Biotechnology Information no. number NPL nonparametric linkage OC osteochondrosis OCD osteochondrosis dissecans OMIM online mendelian inheritance in man PCR polymerase chain reaction POS position QTL quantitative trait locus R reverse RET retention frequency RH radiation hybrid RZPD Resource Center/Primary Database, Berlin SSC sus scrofa chromosome STS sequence-tagged site T thymine TAMU Texas A & M University TBE tris-borate-ethylenediamine tetraacetic acid TE tris-ethylenediamine tetraacetic acid

Ta annealing temperature UV ultraviolet X-Gal 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside

Chapter 1

Introduction

Introduction

Introduction

The horse gene map was - compared with other farm animals - still in its infancy at the beginning of this work, but it has quickly developed during the last few years. Today, genome investigation in horses is aimed at the decoding of the whole horse genome, as it has taken place for humans and several farm animals. Since the horse has not only been used as a helpful worker during the last centuries, but has developed into a dependable partner in equitation today, it is not surprising that the bone and cartilage metabolism and affected diseases like osteochondrosis and navicular disease play an interesting role in horse breeding in the meantime. Osteochondrosis is a developmental orthopaedic disorder of growing animals. The disease is accompanied by abnormal cartilage development in the joints of young growing horses causing subchondral fractures, cysts, wear lines, chondromalacia or cartilage flaps. It can also be manifested in the form of joint mice or free joint bodies (chips) and is known as osteochondrosis dissecans (OCD). A number of articulations can be affected, like the fetlock, hock, carpal, stifle, elbow, hip or vertebral joints. The accumulation of the disease in several horse breeds and families (prevalence in Warmblood and trotter horses between 10 and 79.5%) provides an indication of involved hereditary factors, but responsible genes still have not been found until today. Moreover, nutritional and endocrinological factors, skeletal growth rate and biomechanical trauma seem to play an important role in Osteochondrosis; so the disease appears to be multifactorial. To contribute new results to the comparative human-equine gene map, altogether 17 candidate genes have been chosen. The objective of the present study is the physical mapping of these candidate genes with the previously unknown localization on the horse genome for the first time. Ten genes have been picked due to their possible influence on osteochondrosis, five genes from different solute carrier families, one gene as a candidate gene for pastern dermatitis. This work describes the cytogenetical mapping of the candidate genes by FISH and the confirmation of the localizations by radiation hybrid mapping and offers new findings, which are partially surprising, in the end.

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Overview of chapter contents The presented work consists of eleven chapters of which chapters 3-7 represent already published articles. Chapter 2 reviews the development and the actual situation of equine gene mapping including physical gene maps, comparative gene maps, RH-maps, linkage maps and describing the inheritance of coat colours and several hereditary diseases in horses. Chapters 3-8 describe the physical mapping of 16 genes on the horse genome for the first time. In Chapter 8 and 9 the mapped genes that have not yet been published and the mapping results are concluded and discussed. Chapter 10 provides conclusions recapitulating chapters 3 to 9 and a general discussion. In Chapter 11 the whole work is summarized concisely, while Chapter 12 is a detailed German summary. All tables and figures belonging to the different chapters can be found at the end of this work in the Appendix.

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Chapter 2

Mapping the horse genome and its impact on equine genomics for identification of genes for monogenic and complex traits

D. Stübs and O. Distl

Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Foundation, Germany

Accepted for publication In press: Archives for Animal Breeding

Mapping the horse genome and its impact on equine genomics for identification of genes for monogenic and complex traits

Mapping the horse genome and its impact on equine genomics for identification of genes for monogenic and complex traits

Abstract Since the beginning of investigation in the horse genome in the early nineties, there has been a great progress, especially during the last five years. At the beginning the exploration of monogenic hereditary diseases was one of the main aims, and the causal mutations of several diseases in the horse have been unravelled. The inheritance of coat colours has been explored very detailed, and there exist gene tests for different coat colours such as for chestnut, sabino and tobiano spotting, silver dapple and cream dilution. Information about coat colours and inherited diseases is very important for the breeders and helps avoiding the appearance of lethal genetic factors or undesirable diseases. The most important achievements of horse genome analysis were well-developed linkage, radiation hybrid and cytogenetic genome maps including more than 2950 loci. These maps support comparative analysis of equine hereditary diseases. The present known gene mutations for five diseases in horses have human homologs. Studies on multifactorial diseases such as osteochondrosis and navicular bone disease and on fertility and temperament are underway. At the moment, the whole equine genome is sequenced as it has been done for the human genome and also for other animal genomes. Horse breeding will greatly benefit from identification of QTL for multifactorial traits and gene mutations for congenital anomalies, diseases and performance traits.

Key words: Horse, genome, equine maps, coat colours, inherited diseases

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Zusammenfassung Titel der Arbeit: Kartierung des Pferdegenoms und Genomanalyse von monogenen und komplexen Merkmalen beim Pferd Seit den ersten Arbeiten zur Aufklärung von Genen beim Pferd in den 90er Jahren wurden große Fortschritte in der Genomanalyse beim Pferd gemacht. Für fünf monogen vererbte Krankheiten konnten die verantwortlichen Genmutationen aufgeklärt werden. Die Vererbung der Fellfarben auf populationsgenetischer Ebene ist beim Pferd weitgehend geklärt. Für einige Fellfarben, wie die Fuchsfarbe, die Sabino- und Tobiano-Scheckung, den Silbergenort und Aufhellungsfaktor (Cream Allel) am Albino Genort, konnten Gentests entwickelt werden. Diese Erkenntnisse über die Farballele sind für die Pferdezucht wichtig, und Gentests für die Erkennung von Anlageträgern helfen, das Auftreten von Erbfehlern und Krankheiten zu vermeiden. Die bisher wichtigsten Fortschritte in der Genomanalyse waren die Entwicklung von genetischen, zytogenetischen und Radiation Hybrid Karten, damit Genorte für monogen und multifaktoriell vererbte Merkmale (QTL) kartiert und anschließend vergleichend analysiert werden konnten. Bisher wurden mehr als 2950 Genloci auf diesen Karten kartiert. Arbeiten zu multifaktoriellen Krankheiten wie Osteochondrose und Podotrochlose sowie zur Fruchtbarkeit sowie zu dem Temperament sind auf dem Weg. Momentan wird das Pferdegenom komplett sequenziert wie dies bereits für den Menschen und andere Haustiere durchgeführt wurde. Die Pferdezucht wird davon für die Identifizierung von QTL und Aufklärung von Genmutationen für angeborene Anomalien, Krankheiten und Leistungsmerkmale erheblich profitieren.

Schlüsselwörter: Pferd, Genom, Genkarten, Fellfarben, erbliche Krankheiten

Introduction The first animal genome sequence had been completed already when scientists decoded the sequence of the nematode C. elegans (BETHESDA, 1998). The exploration of mammalian genomes, especially in farm animals, proceeded very fast, and there are several genomes of mammalian animals completed today (FADIEL et

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al., 2005). Genome sequencing in other domestic animals, particularly in pets, forged ahead in the last few years, too, because it was a matter of particular interest for breeders. The chicken, dog, and cow genome have been wholly sequenced and work has started in the pig and horse (BETHESDA, 2004). Having the information on the complete genome sequence is the key on understanding physiological and pathological processes and may revolutionize health research. While scientific investigations had to be confined mainly to the analysis of phenotypic data and pedigrees for familial diseases, today techniques include fluorescence in situ hybridisation (JOHN et al., 1969), somatic cell hybrid panels and radiation hybrid panels (STRACHAN and READ, 1996), comparative mapping

(O`BRIEN and GRAVES, 1991), sequence and mutation analysis, gene expression using microarrays or quantitative real time techniques. New information about mapped genes or markers is shortly posted on one of the databases in the internet and is free available (HU et al., 2001). The progress in investigation of the horse genome especially in the last ten years is the result of concentrated and well-organized scientific work all over the world: 1995 was the year of the foundation of the International Equine Gene Mapping Workshop. Since 1997 this foundation works together with the Horse Technical committee of the National Research Sponsored Project-8 (NRSP-8) of the United States Department of Agriculture National Animal Genome Research Program (USDA-NAGRP) with the aim to decipher the horse genome as soon as possible.

Organization of the horse genome The diploid genome of the domestic horse (Equus caballus) consists of 64 chromosomes, and 13 pairs of the autosomes are biarmed, while the other 18 pairs of the autosomes are acrocentric. The X chromosome is large and metacentric, the Y chromosome small and submetacentric. It is remarkable that the equus przewalski genome consists of 66 chromosomes and that 12 pairs of its autosomes are metacentric and 20 pairs are acrocentric. One pair of the horses` acrocentric chromosomes (ECA5) contains two of the przewalski`s chromosomes (EPR23 and

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EPR24), hence it is possible to mate przewalski and domestic horse and to produce fertile offsprings (AHRENS and STRANZINGER, 2005).

Cytogenetic map Physical identification of loci of interest can be achieved by fluorescence in situ hybridisation (FISH). The first fluorescence in situ hybridisation in the horse was reported by OAKENFULL et al. (1993) with the mapping of the alpha-globin gene complex to horse chromosome (ECA) 13. BREEN et al. (1997) localized the first equine markers when they mapped 18 microsatellites. RAUDSEPP et al. (1997) cytogenetically mapped genes to the genome of the donkey. Lear et al. (2001), RAUDSEPP et al. (2002), GODARD et al. (2000) used goat BAC clones to localize 44 coding sequences on the equine FISH map. LINDGREN et al. (2001) localized another 13 genes by FISH and somatic cell hybrids. One of the first most extensive FISH-map included 136 genes (MILENKOVIC et al. 2002), and to this cytogenetic map further 165 genes were added by FISH and/or radiation hybrid (RH) mapping (PERROCHEAU et al. 2005). The equine gene map now contains 713 genes, 441 on the RH map, 511 on the cytogenetic and 238 on both maps. Several of gene groups attracted special interest for mapping in different animals, such as immunity-related genes: GUSTAFSON et al. (2003) constructed an ordered BAC contig map of the equine major histocompatibility complex (MHC) and TALLMADGE et al. (2003) mapped the ß2-microglobulin gene that is responsible for the light chain of the MHC to ECA1q23-q25. MUSILOVA et al. (2005) mapped another 19 horse immunity-related loci by FISH. NERGADZE et al. (2006) localized the IL8 gene on ECA3q14. Others mapped genes related to the bone-, cartilage- and collagene-metabolism. Genes eventually responsible for bone- or collagene-related hereditary diseases in the horse were mapped by HILLYER et al. (2005), MÜLLER et al. (2005), BÖNEKER et al. (2005) and DIERKS et al. (2006).

Synteny and radiation hybrid maps Synteny mapping and radiation hybrid mapping in the horse work analogously to those in humans, but the utilization of these techniques began more than 20 years

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later than in humans. While there had been five synteny panels in the horse since 1992 (LEAR et al., 1992; WILLIAMS et al., 1993; BAILEY et al., 1995; RANEY et al., 1998), the most important one was the UC Davis panel generated by SHIUE et al. (1999). It included 450 markers and represents the origin for the developed horse- human comparative maps (CAETANO et al., 1999). A 3000-rad panel was developed by KIGUWA et al. (2000). Preliminary comparative maps of horse chromosomes 1 to10 were created using this panel. A great progress was the more extensive 5000-rad whole-genome-radiation hybrid panel that was constructed using 92 horse x hamster hybrid cell lines and 730 equine markers (type I and type II markers) (CHOWDHARY et al., 2003). This panel was one of the most essential tools to construct high-resolution and comparative maps for ECA11 (CHOWDHARY et al. 2003), ECAX (RAUDSEPP et al., 2004), ECA17 (LEE et al., 2004), ECA22 (GUSTAFSON-SEABURY et al. 2005), ECA4 (DIERKS et al., 2006), equine homologs of HSA19 on ECA17, ECA10 and ECA21 (BRINKMEYER- LANGFORD et al., 2005) and equine homologs of HSA2 on ECA6p, ECA15 and ECA18 (WAGNER et al., 2006).

Comparative map Comparative maps between horse and human and horse and mouse are the most important ones. There also exist comparative maps between horse and other mammalians e.g., between horse and donkey (RAUDSEPP et al., 1997, 1999, 2001; RAUDSEPP and CHOWDHARY, 2001), horse and mountain zebra (RAUDSEPP et al., 2002) and E. caballus and E. przewalski (MYKA et al., 2003). Comparative chromosomal painting (Zoo-FISH) helps identifying conserved chromosomal segments among species. Reciprocal painting of chromosomes and segments was performed to compare the horse genome with that of the donkey (RAUDSEPP et al., 1996; CHOWDHARY et al., 1998; YANG et al., 2004). One approach to localize genes and to expand the comparative gene map between horse and man is to pick genes or DNA-/RNA-sequences from genes physically mapped and cloned in humans for screening filters of a genomic horse BAC library. After verification of the selected gene sequence on the genomic equine BAC clone,

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FISH is used for mapping this BAC clone on horse metaphase spreads. There are three equine genomic BAC-libraries available: the equine BAC library of the Chidren`s Hospital of Oakland Research Institute, CHORI-241 (GODARD et al., 1998), one generated by the Texas A&M University (TAMU) and the other by the Institut de la National Recherche Agronomique (INRA-LGBC library) (CHOWDHARY et al., 2003). CAETANO et al. (1999) published an extensive comparative map between horse and man containing 68 equine type I loci. The most topical comparative map between horse, donkey and human was generated by cross-species painting (YANG et al., 2004). The human-donkey map was the first genome-wide comparative map between these species generated by chromosome painting. This horse-human comparative map agrees basically with the comparative map of CHOWDHARY et al. (2003), except from varying results especially for ECA1. For several horse chromosomes, detailed comparative maps have been developed. A detailed physical map of the equine Y-chromosome was generated (RAUDSEPP et al., 2004) and compared to the human and other mammalian species with the result that the equine Y-chromosome shows the most homology with the Y-chromosome of the pig. A comparative RH map for ECA17 was developed by LEE et al. (2004), and there exists a detailed RH map of ECA22 comparing the equine loci to human, dog, cat, bovids, elephant and bat, rodents, dolphins and other mammalians (GUSTAFSON- SEABURY et al., 2005). This map covered estimated 64 Mb of physical length of chromosome 22 (831 cR) and the 83 markers had an average distance of 10 cR. It was remarkable, that the most common configuration for the ECA22 homologs consisted for Hartmann’s zebra as expected, but also for carnivores, hippos, primates, rabbits, squirrels and bats. The conserved synteny between the compared animals can be taken as an indicator of the degree of putative ancestral relationship between the different species. BRINKMEYER-LANGFORD et al. (2005) compared equine homologs of HSA19 to other mammals using 89 loci for the 5000-rad-RH- panel and four loci for FISH. The homologs to HSA19 were localized on ECA7, 21 and 10. This comparison demonstrated an overview of the evolution of putative ancestral HSA19 chromosomal segments in more than 50 mammalian species.

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PERROCHEAU et al. (2006) developed a medium density horse gene map using 323 genes evenly distributed over the human genome. They mapped 87 genes by FISH and 186 genes by the equine 5000-rad RH panel and thereby detected previously unknown homology between ECA27 and HSA8 as well as between ECA12p and HSA11p.

Linkage map Genetic and physical assignment of equine microsatellites began almost ten years ago. BREEN et al. (1997) isolated 20 equine microsatellites from a genomic phage library and mapped them by linkage mapping and FISH. GODARD et al. (1997) assigned 36 new horse microsatellites, 11 of them from plasmid libraries and 25 from a cosmid library. The first equine linkage map included 140 markers (LINDGREN et al., 1998) and a more extensive linkage map consisted of 161 loci in 29 linkage groups for 26 autosomes (GUERIN et al., 1999). The first map covering all autosomes and the sex- chromosomes using 353 microsatellite-markers was generated by SWINBURNE et al. (2000). The microsatellites were mapped to 42 linkage groups and covered 1780 cM of the horse genome. A second generation linkage map was published by the International Equine Gene Mapping Workshop (GUERIN et al. 2003). This linkage map was based on testing 503 half-sibling offspring from 13 sire families. The map included 344 markers in 34 linkage groups representing all 31 autosomes, but neither the X- nor the Y- chromosome. The linkage groups covered 2262 cM with an average interval between loci of 10.1 cM, ranging from 0 to 38.4. Another 61 new horse microsatellite loci were assigned by SWINBURNE et al. (2003). TOZAKI et al. (2004) reported the characterization of 341 newly isolated microsatellite markers of which 256 were assigned to equine chromosomes. PENEDO et al. (2005) added 359 microsatellites to the second generation linkage map. Alltogether, this linkage map consisted of 766 markers assigned to the 30 autosomes and the X-chromosome spanning 3740 cM with an average marker density of 6.3 cM. SWINBURNE et al. (2006) generated a linkage map including 742

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markers in 32 linkage groups according to the 31 autosomes and the X-chromosome. All linkage groups together span 2772 cM with an average interval of 3.7 cM between the markers.

Chromosomal abnormalities There exist several chromosomal abnormalities in the horse. Of great interest and therefore well analyzed are those concerning the sex-chromosomes and influencing the fertility of mares: The most common abnormality in mares, X0 Gonadal Dysgenesis (X monosomy), is characterised by the lack of one X-chromosome (63,X0) and was primarily described by HUGHES et al. (1975). This defect was accompanied with infertility of the affected mare. POWER (1986) reported another abnormality in horses, the XY Gonadal Dysgenesis. The affected horses` phenotype is female, but their genotype is 64,XY leading to infertility (BOWLING et al., 1987). There exist also infertile mares with the genotype 65,XXX (CHANDLEY et al., 1975). KAKOI et al. (2005) published a statistical evaluation of sex chromosome abnormalities and analyzed data of 17471 light-breed foals with the result that 0.15 % of the analyzed population showed an XO-, 0.02% an XXY- (and/ or mosaics/chimaeras) and 0.01% an XXX-genotype. Unlike the sex chromosomes, chromosomal abnormalities of the autosomes are rarely encountered. Cases of trisomy are similar to human Downs and associated with mental retardation failure to thrive (LEAR et al., 1999).

Coat colours There exist several genetically characterized major genes that influence the coat colour of a horse: Agouti-gene, Extension-gene, Cream dilution gene, Tobiano-gene, White-gene, Greying hair-gene, Dun-gene, Roan-gene, Sabino-gene, Silver-gene, and the Leopard Complex-gene. Knowledge about the inheritance of coat colours is very important for the breeders, so it is comparatively well explorated and for several genes exist gene tests, too. Chromosomal locations and mutations of genes associated with equine coat colours are summarized in Table.

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The White-gene and the Greying hair-gene can mask all the other coat colour genes, so they are mentioned first. White-gene (W-gene) and Sabino spotting Horses with the dominant allele “W” lack pigment in skin and hair, so their coat colour is white at birth, but eyes are dark or sometimes blue. The W-mutation was mapped to a region of ECA3q22 where the equine KIT gene is located (MAU et al., 2004). The homozygous “WW” is lethal at a very early stage of pregnancy as it is in mice, too. Since the action of the dominant white allele seems not always to be fully penetrant, some horses homozygously “WW” are born alive with the phenotype of sabino spotting. All non-white horses are homozygous recessive “ww”. In contrast to the greying gene, horses with the dominant “W” are not able to produce any pigment, white horses with the “G”-gene instead can produce pigment but lose their pigment with aging. Sabino pattern is characterized by irregulary bordered white patches on the lower limbs and face and often includes belly and interspersed white hairs on the midsection. Sabino spotting is found in many light horse breeds such as Tennessee walking horse, Missouri Foxtrotter, Shetland pony and American Paint Horse. A single nucleotide exchange in the 3’-splice site of intron 16 of the equine KIT gene causes a partial skipping of exon 17 and the Sabino spotting (BROOKS and BAILEY, 2005). Furthermore, close association between KIT polymorphisms and roan coat colour was detected in horses (MARKLUND et al., 1999). Greying-gene (G-gene) The dominant allele “G” is responsible for progressive greying in hairs. If a foal that possesses the allele “G” is born coloured it will progressively turn white as an aged animal. The Grey-locus was mapped to ECA25 by SWINBURNE et al. (2002) and by HENNER et al. (2002). The mapping of the G-gene was of particular importance because the Grey-gene and the appearance of melanomas in horses are associated: grey horses get melanomas with a frequency of at least 80% in horses older than 10 years whereas melanomas are rare in non-grey horses. SWINBURNE et al. (2002)

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localized the G-locus 13 cM proximal of the marker TKY316 and 17 cM distal of the marker UCDEQ464 using a whole genome scan. PIELBERG et al. (2005) refined the grey-locus to a region smaller than 6.9 Mb on ECA25 using comparative mapping to mouse and human. In this refined region, no obvious candidate genes for pigmentation disorders could be identified. Several possible candidate genes responsible for different coat colours including agouti- signaling-protein (ASIP), melanocortin-1-receptor (MC1R), tyrosinase-related protein 1 (TYRP1) and silver homolog (SILV, PMEL17) had been excluded before (RIEDER et al., 2001; LOCKE et al., 2002). Agouti-gene (A-gene) The Agouti locus with the dominant “A” and recessive “a” allele is responsible for the distribution pattern of black hair. The interaction with the extension-gene is responsible if the horse is bay or black: A horse that is homozygous recessive “aa” will be black if it also possesses a dominant “E” allele. If a horse possesses at least one “A” allele together with the “E” allele, it will be bay. However, if a horse is homozygous recessive “ee”, it will look sorrel or chestnut, irrespective if it carries an “A” or “a”. This is important for the breeders, because it is possible that a sorrel or chestnut horse can also produce blacks. RIEDER et al. (2001) characterized equine MC1R (melanocortin-1-receptor) and ASIP (agouti signaling protein), and completed a partial sequence of TYRP1. They detected an 11-bp deletion in exon 2 (ADEx2) of ASIP which was completely associated with horse recessive black coat color in horses of 9 different breeds. The frameshift initiated by ADEx2 was supposed to act as a loss-of-function ASIP mutation. Extension gene (E-gene) The Extension locus controls the expression of black pigment in horses. The equine melanocyte stimulating hormone receptor (MC1R) on ECA3 encodes the Extension locus (MARKLUND et al., 1996). A missense mutation in MC1R was associated with light colour, whereas the wildtype allele corresponded to dark pigmentation. A black or bay horse possesses at least one “E” allele. A horse that is homozygous “ee” is either chestnut or sorrel. For horse breeders where black colour is valued, it is

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important, if a horse is homozygous “EE”, because that means that it will never have a chestnut or sorrel foal. Cream dilution gene (C-gene) The dominant “C” allele causes a reduction in red pigment in hairs. A sorrel horse inheriting one “C” allele is palomino (ee/Cc), a sorrel inheriting “C/C” is cremello (ee/CC). A bay horse inheriting one “C” allele is buckskin (E-/Cc) and a horse homozygous for “C” is a perlino (E-/CC). It is also possible that grey or dun horses carry the “C” allel. The C-locus was localized to ECA21 and TYR (tyrosinase) could be excluded as candidate for the cream dilution gene (Locke et al. 2001). MARIAT et al. (2003) identified the causal mutation similarly to mice and humans in exon 2 of the membrane associated transporter protein (MATP) gene mapping to ECA21p17. A gene test based on this G to A transition could be developed to control if a horse carries a dominant “C” allele. Tobiano-gene (To-gene) The “To” allele is dominantly inherited and is responsible for white spotting characterized by distinct borders crossing the dorsal midline and including at least one if not all four limbs. There was identified a MSPI polymorphism for the Tobiano- locus in intron 13 of the equine homologue of the proto-oncogene c-kit (KIT) gene (KM1 locus) which is strongly associated with the Tobiano gene but not causative (BROOKS et al., 2002). If a horse does not possess the Tobiano-allele, the test result will be designated KM0/KM0. If a horse carries one “To” allele, it will be tested KM0/KM1, and if the test is KM1/KM1, the horse is probably a homozygous Tobiano. However, solid-coloured horses can also possess the KM1 allele. This test is useful for breeders valuing homozygous Tobianos, because they will always produce spotted foals. Silver-gene (Z-gene) A mutation in the Silver (PMEL17) gene had recently been identified that produces in black horses a chocolate coat colour with flaxen mane and tail and in bay horses lightened coat colour in lower legs and flaxen mane and tail (BRUNBERG et al., 2006). This mutation has no effect on chestnut-coloured horses. A missense mutation in exon 11 changing arginine to cysteine (Arg618Cys) was likely to be

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causative as complete association with the Silver phenotype was observed across several horse breeds. A further silent mutation in intron 9 was also completely associated with the Silver phenotype. Appaloosa coat colour TERRY et al. (2004) investigated the appaloosa coat colour which is characterized by several different spotting patterns ranging from a few white specks on the rump to an almost completely white animal. They excluded the KIT gene as possible candidate gene causing Rw colour pattern in mice and also the candidate genes microphthalmia-associated transcription factor (MITF) and mast cell growth factor (MGF) (TERRY et al., 2001, 2002). They found that the autosomal incompletely dominant gene LP (leopard complex) responsible for the appaloosa coat pattern in horses mapped to ECA1q (TERRY et al., 2004).

Table Molecular genetic characterization of coat colour loci in horse (Molekulargenetische Charakterisierung von Farbgenorten beim Pferd) Locus Gene ECA Mutation Reference Agouti ASIP 22q15-q16 11 bp deletion RIEDER et al. (2001) Extension MC1R 3p12 C>T missense MARKLUND et al. (1996) mutation Cream MATP 21p17 G>A missense MARIAT et al. (2003) mutation Sabino KIT 3q22 T>A mutation BROOKS and BAILEY (splice (2005) mutation) Tobiano KIT 3q22 C>G missense BROOKS et al. (2002) mutation Silver PMEL17 6q23 C>T missense BRUNBERG et al. (2006) mutation ECA: Equus caballus autosome.

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Equine genetic diseases and the development of gene tests The most important information breeders take from gene investigation are probably new results as to diseases or phenotypic features. While inheritance of colours is usually comparatively simple with different alleles that are either dominant or recessive responsible for all colours, investigation of inheritance of diseases is often much more complicated, for example multifactorial and influenced by lots of different genes. There are 188 diseases reported in the horse that are discussed to be inherited. For 20 well-explorated diseases only a single locus is responsible for the appearance. This is probably one of the reasons for the fact that there are only a few gene tests available for important diseases but for many of the possibly inherited diseases in the horse the inheritance is still unknown.

Monogenic equine diseases Lethal White Foal Syndrome (LWFS) The Lethal White Foal Syndrome is observed in horse breeds with white coat spotting patterns called overo, e.g., in Paints and Pintos. Affected foals are almost completely white and die shortly after birth due to severe intestinal blockage resulting from a lack of nerve cells in the distal portion of the large intestine (aganglionic megacolon). The disease is similar to Hirschsprung disease in humans. As the Hirschsprung disease is caused by a mutation in the endothelian B receptor gene (EDNRB), METALLINOS et al. (1998) investigated the influence of the EDNRB in horses with Lethal White Foal Syndrome and found an association between a missense mutation in the EDNRB-gene and the appearance of LWFS like in humans for the Hirschsprung disease. YANG et al. (1998) could show that a dinucleotide TC>AG mutation was associated with LWFS when homozygous and with the overo phenotype when heterozygous. Herlitz junctional epidermolysis bullosa (epitheliogenesis imperfecta) The pathological signs of epitheliogenesis imperfecta closely match a similar disease in humans known as Herlitz junctional epidermolysis bullosa, which is caused by a mutation in one of the genes (LAMA3, LAMB3 and LAMC2) coding for the subunits of

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the laminin 5 protein. Herlitz junctional epidermolysis bullosa (HJEB) is a lethal disease that causes blistering of the skin and mouth epithelia, and sloughing of hooves in newborn foals, especially in American Saddlebred horses and Belgian draft horses (JOHNSON et al., 1998). Pedigree studies demonstrated that the trait followed a monogenic autosomal recessive inheritance pattern and a genetic mapping study revealed that HJEB in American Saddlebred horses was linked to microsatellites on ECA8q where LAMA3 is also located (LIETO and COTHRAN, 2003). SPIRITO et al. (2002) detected a cytosine insertion in exon 10 in the LAMC2 gene as being responsible for HJEB in Belgian draft horses and BAIRD et al. (2003) developed a gene test that works with hair of the horses and is now available for owners of registered horses. The same mutation was found to be associated with the same condition in the French draft horse breeds, Trait Breton and Trait Comtois (MILENKOVIC et al., 2003). Hyperkalemic periodic paralysis Hyperkalemic periodic paralysis (HYPP) is an inherited disease of the muscles that causes attacks of paralysis that can lead to sudden death that especially affects American Quarter horses. It is inherited as an autosomal codominant genetic defect so that heterozygous horses are affected, too (ZEILMANN, 1993). HYPP is the result of a missense mutation of the gene encoding the a-chain of the adult muscle sodium channel. RUDOLPH et al. (1992) described the point mutation in transmembrane domain IVS3 that is responsible for the occurrence of the disease. Even though the attacks can be treated with azetazolamide (KOLLIAS-BAKER, 1999) and the disease will not always lead to death, it is useful to test horses if they are carriers or not. Severe Combined Immunodeficiency (SCID) SCID is an inherited disease known in different species that always causes an early death of affected animals because of the incapability to generate antigene-specific immune responses; there exists a comparative study between the different mechanisms that lead to SCID in humans, mice, horses and dogs (PERRYMAN, 2004). SCID is recessively inherited in Arabian horses. Horses affected with SCID cannot react in a natural way of protection against diseases. They do not develop the necessary active protection reactions because they lack intact lymphocytes. Due to

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this reason, affected foals will not become older than six months. SHIN et al. (1997) tested candidate genes from the mouse and could show that SCID is caused by a frameshift mutation in the gene for DNA-dependent protein kinase catalytic subunit (DNA-PK) mapping to horse chromosome 9. This mutation results in the lack of a full- length kinase. Glycogen Branching Enzyme Deficiency (GBED) GBED is a glycogene storage disease similar to the human glycogen storage disease type IV. The disease is lethal for the affected foals with a great variability of symptoms ranging from abort or stillbirth until being euthanized due to weakness. VALBERG et al. (2001) described the disease in American Quarter horses. Common to GBED are the accumulation of unbranched polysaccharides in tissues and a profound decrease of glycogen branching enzyme activity in cardiac and skeletal muscle as well as in liver and peripheral blood cells of affected foals. The pedigree analysis supported an autosomal recessive mode of inheritance. WARD et al. (2003) mapped the GBE1 gene to ECA26q12-q13, and later on, detected the C- to A-mutation at base 102 of the GBE1 gene causing the disease (WARD et al., 2004). An autosomal recessive mode of inheritance was confirmed. It is now possible to test horses whether they are carriers of the GBE1 allele.

Genetically complex equine diseases Osteochondrosis (OC) Osteochondrosis (OC) is a developmental orthopaedic disorder frequently observed in young horses (VAN DE LEST et al., 1999). Signs of osteochondrosis are lesions of the cartilage in the joints like subchondral fractures, subchondral cysts, wear lines, chondromalacia, cartilage flaps and joint mice or free joint bodies (chips). Hereditary factors play an important part in the pathogenesis of OC. An optimized microsatellite marker set for complete genome scans in horses was developed including 155 highly polymorphic markers equally distributed at a distance of about 20 cM. Data from 14 half-sib families of Hanoverian Warmblood horses were analysed using this marker set to detect quantitative trait loci (QTL) with significant influence on the development of OC (BÖNEKER et al., 2006; DIERKS and DISTL, 2006). QTL for osteochondrosis

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in fetlock and hock joints were found on horse chromosomes 2, 3, 4, 5, 15, 16, 19, and 21. In a population of South German coldblood horses a further whole genome scan for OC in fetlock and hock joints was carried out (WITTWER et al., 2006; WITTWER and DISTL, 2006). In total, 17 chromosome-wide significant QTL were found on different equine chromosomes with influence on the development of OC in fetlock and hock joints. Navicular disease Navicular disease (navicular syndrome or podotrochlosis) is a chronic and usually progressive, degenerative alteration of the equine podotrochlea (RIJKENHUIZEN, 1989). Pathological alterations can primarily affect the navicular bone (os sesamoideum distale), the navicular bursa (bursa podotrochlearis) or the distal end of the deep digital flexor tendon. The disease may be part of the osteoarthritis complex (SVALASTOGA and SMITH, 1983). A microsatellite marker set to be applied in Hanoverian warmblood horses for a whole genome scan was prepared and then a whole genome scan in 144 descendants of 17 Hanoverian warmblood stallions was performed. The genotyped horses were randomly sampled from the whole Hanoverian warmblood breeding district. QTL on different horse chromosomes were identified such as on ECA2, 3, 4, and 10 (DIESTERBECK et al., 2006). According to SVALASTOGA and SMITH (1983) increased bone marrow pressure and lengthened contrast passage indicate similarities between osteoarthritis (OA) in humans and navicular disease in horses. About 50 different positional candidate genes have been reported for OA in humans. These candidate genes encode different types of collagens, hormone receptors and interleukin receptors, growth factors and metalloproteinases. About 13,966 equine cartilage expressed sequence tags (ESTs) and further 23,171 ESTs from other cDNA libraries as well as BAC end sequences or whole genome sequences can be used for identification of single nucleotide polymorphisms in functional and positional candidate genes. However, since navicular disease does not occur in humans, it is not clear whether genes for OA are suitable candidates for navicular bone disease in horses.

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Temperament Temperament is considered important in horses as optimal performance is related with a good temperament. The neurotransmitter-related dopamine D4 receptor (DRD4) gene harbours many polymorphisms in the coding region and among them, the variable number of tandem repeats polymorphism consisting of a 48-bp repeat unit was shown to be associated with novelty-seeking in humans (BENJAMIN et al., 1996; EBSTEIN et al., 1996). In horses, this part of DRD4 included repeats of an 18- bp repeat unit (HASEGAWA et al., 2002). Two temperament scores, curiosity and vigilance were associated with a single nucleotide polymorphism (A>G substitution) of DRD4 but no difference in the number of repeats among the 136 two-year-old thoroughbred horses was found (MOMOZAWA et al., 2005). Horses without the A allele had higher curiosity and lower vigilance scores than those with the A allele. Polymorphisms of the serotonin transporter (SLC6A4) gene were screened for associations with an anxiety score in 67 Thoroughbred horses (MOMOZAWA et al., 2006). However, no significant associations could be identified in this study.

Stallion fertility Seminal plasma proteins that are produced by the epididymis and the accessory glands play an important role in sperm maturation. Some of these proteins bind to spermatozoa and act as binding partners in the female genital tract and others are required to suppress adverse immune reactions directed against spermatozoa. Among the seminal plasma proteins, cystein-rich secretory protein 3 (CRISP3) is found in large amounts in the seminal plasma of stallions (SCHAMBONY et al., 1998). Association studies for CRISP3 polymorphisms in 107 Hanoverian stallions revealed a significant influence on fertility at artificial insemination of mares (HAMANN et al., 2006). Stallions being heterozygous for an E208K mutation within the CRISP3 gene had less insemination success than those without this mutation or homozygous for this mutation.

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Conclusions and perspectives There has been great progress in the horse genome investigation especially during the last ten years, but compared to the human genome or to animals like the mouse, the horse gene map still shows many unexplored areas. For the coat colours, the inheritance of the major colours is already resolved, but for several possibly inherited diseases the mode of inheritance is still not detected despite of intensive investigation in the last years, as for example for the appearance of “parrot mouth”, “hyperelastosis cutis”, “laminitis”, “osteochondrosis” and many more. It is always difficult if there are no suitable candidate genes from the human because no equivalent diseases exist in human. Another problem is that many diseases are not only characterized by a single mutation or a simple dominant or recessive mode of inheritance but are multifactorial and many different genes or environmental effects like feeding or movement influence the appearance of these diseases. The ongoing refinement of the comparative map between horse and human plays an important role in genomic studies in the horse: Without the generation of tools like the 5000-rad-RH-panel by CHOWDHARY et al. (2003) it would not be possible to map new loci as accurately as they can be localized today. Laborious methods like the fluorescence in-situ hybridization (FISH) are increasingly eclipsed by methods that are more timesaving and that lead to more new results in one experiment, like the radiation hybrid panel mapping (RH). Horse genome sequencing, funded by the National Human Genome Research Institute, had started in February 2006 and the sequencing of the about 2.7 billion DNA bases is nearly complete. A whole genome shotgun approach is being used for sequencing a female thoroughbred named “Twilight”. A high-quality genome sequence of Equus caballus will be elaborated together with an analysis of sequence variation of 100,000 randomly chosen DNA segments from mares of seven additional breeds. Breeds selected for this single nucleotide polymorphisms (SNPs) analysis included thoroughbred, Quarterhorse, Arabian horse, American Standardbred pacer, Akalteke, Andalusian and Icelandic horse. More than one million SNPs should be identified. The horse genome project has emerged from an ongoing effort to highlight

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the most evolutionarily conserved segments of the human genome by identifying genetic similarities between several different mammalian genomes and human genome. A high-quality horse genome sequence and the large catalogue of SNPs will be the key to identify the genetic contributions to the aetiopathogenesis of diseases, physiological and pathological processes and to the athletic performance of horses in sports, agriculture and transportation.

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BAILEY, E.: Mapping 31 horse genes in BACs by FISH; identification of chromosomal rearrangements and conserved synteny relative to the human gene map. Chrom. Res., 9, (2001), 261-262 LEE, E.J.; RAUDSEPP, T.; KATA, S.R.; ADELSON, D.; WOMACK, J.E.; SKOW, L.C.; CHOWDHARY, B.P.: A 1.4-MB interval RH map of horse chromosome 17 provides detailed comparison with human and mouse homologues. Genomics, 83, (2004), 203-215

LIETO, L.D.; COTHRAN, E.G.: The epitheliogenesis imperfecta locus maps to equine chromosome 8 in American Saddlebred horses. Cytogenet. Genome Res., 102, (2003), 207-210 LINDGREN, G.; SANDBERG, K.; PERSSON, H.; MARKLUND, S.; BREEN, M.;

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GUERIN, G.; ELLEGREN, H.; BINNS, M.M.: Physical anchorage and orientation of equine linkage groups by FISH mapping BAC clones containing microsatellite markers. Anim. Genet., 32, (2001), 37-39 LOCKE, M.M.; RUTH, L.S.; MILLON, L.V.; PENEDO, M.C.; MURRAY, J.D.;

BOWLING, A.T.: The cream dilution gene, responsible for the palomino and buckskin coat colours, maps to horse chromosome 21. Anim. Genet., 32, (2001), 340-343 LOCKE, M.M.; PENEDO, M.C.; BRICKER, S.J.; MILLON, L.V.; MURRAY, J.D.: Linkage of the grey coat colour locus to microsatellites on horse chromosome 25. Anim. Genet., 33, (2002), 329-337

MARIAT, D.; TAOURIT, S.; GUERIN, G.: A mutation in the MATP gene causes the cream coat colour in the horse. Genet. Sel. Evol., 35, (2003), 119-135 MARKLUND, L.; MOLLER, M.J.; SANDBERG, K.; ANDERSSON, L.: A missense mutation in the gene for melanocyte-stimulating hormone receptor (MC1R) is associated with the chestnut coat color in horses. Mamm. Genome, 7, (1996), 895-899 MARKLUND, S.: MOLLER, M.; SANDBERG, K.; ANDERSSON, L.: Close association between sequence polymorphism in the KIT gene and the roan coat colour in horses. Mamm. Genome, 10, (1999), 283-288

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MYKA, J.L.; LEAR, T.L.; HOUCK, M.L.; RYDER, O.A.; BAILEY, E.: FISH analysis comparing genome organization in the domestic horse (Equus caballus) to that of the Mongolian wild horse (E. przewalski). Cytogenet. Genome Res., 102, (2003), 222-225 NERGADZE, S.G.; MAGNANI, E.; ATTOLINI, C.; BERTONI, L.; ADELSON, D.L.;

CAPPELLI, K.; VERINI SUPPLIZI, A.; GIULOTTO, E.: Assignment of the equus caballus interleukin 8 gene (IL8) to chromosome 13q14.2 ? 14.3 by in situ hybridization. Cytogenet. Genome Res., 112, (2006), 341B

OAKENFULL, E.A.; BUCKLE, V.J.; CLEGG, J.G.: Localization of the horse (Equus caballus) alpha-globin gene complex to chromosome 13 by fluorescence in situ hybridization. Cytogenet. Cell Genet., 62, (1993), 136-138

O´BRIEN, S.J.; GRAVES, M.J.A.: Report of the committee on comparative gene mapping. Cytogenet. Cell Genet., 58, (1991), 1124-1151 PENEDO, M.C.T.; MILLON, L.V.; BERNOCO, D.; BAILEY, E.; BINNS, M.; CHOLEWINSKI, G.; ELLIS, N.; FLYNN, J.; GRALAK, B.; GUTHRIE, A.; HASEGAWA, T.; LINDGREN, G.; LYONS, L.A.; ROED, K.H.; SWINBURNE, J.E.; TOZAKI, T.: International equine gene mapping workshop report: a comprehensive linkage map constructed with data from new markers and by merging four mapping resources. Cytogenet. Genome Res., 111, (2005), 5-15 PERROCHEAU, M.; BOUTREUX, V.; CHADI-TAOURIT, S.; CHOWDHARY, B.P.; CRIBIU, E.P.; DE JONG, P.J.; DURKIN, K.; HASEGAWA, T.; HIROTA, K.; IANUZZI, L.; INCARNATO, D.; LEAR, T.L.; RAUDSEPP, T.; PERUCATTI, A.; DI

MEO, G.P.; ZHU, B.; GUERIN, G.: Cytogenetic mapping of 150 genes on the horse genome. Plant and Animal Genome XIII Conference, (2005), Januar 15-19, 2005, San Diego, CA

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PERROCHEAU, M.; BOUTREUX, V.; CHADI, S.; MATA, X.; DECAUNES, P.; RAUDSEPP, T.; DURKIN, K.; INCARNATO, D.; IANNUZZI, L.; LEAR, T.L.; HIROTA, K.; HASEGAWA, T.; ZHU, B.; DE JONG, P.; CRIBIU, E.P.;

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CHOWDHARY, B.P.: Comparison of horse chromosome 3 with donkey and human chromosomes by cross-species painting and heterologous FISH mapping. Mamm. Genome, 10, (1999), 277-282

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RAUDSEPP, T.; CHOWDHARY, B.P.: Correspondence of human chromosomes 9, 12, 15, 16, 19 and 20 with donkey chromosomes refines homology between horse and donkey karyotypes. Chrom. Res., 9, (2001), 623-629

RAUDSEPP, T.; MARIAT, D.; GUERIN, G.; CHOWDHARY, B.P.: Comparative FISH mapping of 32 loci reveals new homologous regions between donkey and horse karyotypes. Cytogenet. Genome Res., 94, (2001), 180-185

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H.B.;WOMACK, J.E.; SKOW, L.C.; CHOWDHARY, B.P.: A detailed physical map of the horse Y chromosome. Proc. Natl. Acad. Sci. USA, 101, (2004), 9321-9326

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SCHAMBONY, A.; GENTZEL, M.; WOLFES, H.; RAIDA, M.; NEUMANN, U.; TÖPFER-PETERSEN, E.: Equine CRISP-3: primary structure and expression in the male genital tract. Biochim. Biophys. Acta, 1387, (1998), 206-216 SHIN, E.K.; PERRYMAN, L.E.; MEEK, K.:

Evaluation of a test for identification of Arabian horses heterozygous for the severe combined immunodeficiency trait. J. Am. Med. Assoc., 211, (1997), 1268- 1270 SHIUE, Y.L.; BICKEL, L.A.; CAETANO, A.R.; MILLON, L.V.; CLARK, R.S.; EGGLESTON, M.L.; MICHELMORE, R.; BAILEY, E.; GUERIN, G.; GODARD, S.; MICKELSON, J.R.; VALBERG, S.J.; MURRAY, J.D.; BOWLING, A.T.: A synteny map of the horse genome comprised of 240 microsatellite and RAPD markers. Anim. Genet., 30, (1999), 1-9 SPIRITO, F.; CHARLESWORTH, A.; LINDER, K.; ORTONNE, J.P.; BAIRD, J.; MENEGUZZI, G.: Animal models for skin blistering conditions: absence of laminin 5 causes hereditary junctional mechanobullous disease in the Belgian horse. J. Invest. Dermatol., 119, (2002), 684-691

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BJARNASON, J.; ALLEN, T.; BINNS, M.: First comprehensive low-density horse linkage map based on two 3-generation, full-sibling, cross-bred horse reference families. Genomics, 66, (2000), 123-134

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SWINBURNE, J.E.; HOPKINS, A.; BINNS, M.M.: Assignment of the horse grey coat colour gene to ECA 25 using whole genome scanning. Anim. Genet., 33, (2002), 338-342 SWINBURNE, J.E.; TURNER, A.; ALEXANDER, L.J.; MICKELSON, J.R.; BINNS, M.M.: Characterization and linkage map assignments for 61 new horse microsatellite loci (AHT49-109). Anim. Genet., 34, (2003), 65-68 SWINBURNE, J.E.; BOURSNELL, M.; HILL, G.; PETTITT, L.; ALLEN, T.; CHOWDHARY, B.P.; HASEGAWA, T.; KUROSAWA, M.; LEEB, T.; MASHIMA,

S.; MICKELSON, J.R.; RAUDSEPP, T.; TOZAKI, T.; BINNS, M.: Single linkage group per chromosome genetic linkage map for the horse, based on two three-generation, full-sibling, crossbred horse reference families. Genomics, 87, (2006), 1-29 TALLMADGE, R.L.; LEAR, T.L.; JOHNSON, A.K.; GUERIN, G.; MILLON, L.V.;

CARPENTER, S.L.; ANTCZAK, D.F.: Characterisation of the ß2-microglobulin-gene of the horse. Immunogenet., 54, (2003), 725-733

TERRY, R.R.; BAILEY, E.; BERNOCO, D.; COTHRAN, E.G.: Linked markers exclude KIT as the gene responsible for appaloosa coat colour spotting patterns in horses. Anim. Genet., 32, (2001), 98-101

TERRY, R.B.; BAILEY, E.; LEAR, T.; COTHRAN, E.G.: Rejection of MITF and MGF as the genes responsible for appaloosa coat colour patterns in horses. Anim. Genet., 33, (2002), 82-84 TERRY, R.B.; ARCHER, S.; BROOKS, S.; BERNOCO, D.; BAILEY, E.: Assignment of the appaloosa coat colour gene (LP) to equine chromosome 1. Anim. Genet., 35, (2004), 134-137 TOZAKI, T.; PENEDO, M.C.; OLIVEIRA, R.P.; KATZ, J.P.; MILLON, L.V.; WARD, T.; PETTIGREW, D.C.; BRAULT, L.S.; TOMITA, M.; KUROSAWA, M.; HASEGAWA,

T.; HIROTA, K.: Isolation, characterization and chromosome assignment of 341 newly isolated equine TKY microsatellite markers. Anim. Genet., 35, (2004), 487-496

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FYFE, J.; MICKELSON, J.R.: Glycogen branching enzyme deficiency in quarter horse foals. J. Vet. Intern. Vet., 15, (2001), 572-580 VAN DE LEST, C.H.A.; VAN DEN HOOGEN, B.M.; VAN WEEREN, P.R.; BROUWERS, J.F.H.M.; VAN GOLDE, L.M.G.; BARNEVELD, A.: Changes in bone morphogenic enzymes and lipid composition of equine osteochondrotic subchondral bone. Equine vet. J., Suppl. 31, (1999), 31-37 WAGNER, M.L.; RAUDSEPP, T.; GOH, G.; AGARWALA, R.; SCHÄFFER, A.A.; DRANCHAK, P.K.; BRINKMEYER-LANGFORD, C.; SKOW, L.C.; CHOWDHARY, B.P.; MICKELSON, J.R.: A 1.3-Mb interval map of equine homologs of HSA2. Cytogenet. Genome Res. 112, (2006), 227-234. WARD, T.L.; VALBERG, S.J.; LEAR, T.L.; GUERIN, G.; MILENKOVIC, D.; SWINBURNE, J.E.; BINNS, M.M.; RAUDSEPP, T.; SKOW, L.; CHOWDHARY, B.P.; MICKELSON, J.R.: Genetic mapping of GBE1 and its association with glycogen storage disease IV in American Quarter horses. Cytogenet. Genome Res. 102, (2003), 201-206 WARD, T.L.; VALBERG, S.J.; ADELSON, D.L.; ABBEY, C.A.; BINNS, M.M.;

MICKELSON, J.R.: Glycogen branching enzyme (GBE1) mutation causing equine glycogen storage disease IV. Mamm. Genome 15, (2004), 570-577 WILLIAMS, H.; RICHARDS, C.M.; KONFORTOV, B.A.; MILLER, J.R.; TUCKER,

E.M.: Synteny mapping in the horse using horse-mouse heterohybridomas. Anim. Genet. 24, (1993), 257-260 WITTWER, C.; DISTL, O.: Mapping quantitative trait loci for osteochondrosis in South German Coldblood horses. Proceedings of the 30th International Conference on Animal Genetics, ISAG 2006, 20-25 August 2006, Porto Seguro, Brazil, D330

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WITTWER, C.; LÖHRING, K.; HAMANN, H.; ROSENBERGER, E.; DISTL, O.: Mapping quantitative trait loci for osteochondrosis in South German Coldblood horses. Anim. Genet., (2006), in review YANG, G.C.; CROAKER, D.; ZHANG, A.L.; MANGLICK, P.; CARTMILL, T.; CASS D.: A dinucleotide mutation in the endothelin-B receptor gene is associated with lethal white foal syndrome (LWFS): a horse variant of Hirschsprung disease (HSCR). Hum. Mol. Genet., 7, (1998), 1047-1052 YANG, F.; FU, B.; O`BRIEN, P.C.; NIE, W.; RYDER, O.A.; FERGUSON-SMITH, M.A.: Refined genome-wide comparative map of the domestic horse, donkey and human based on cross-species chromosome painting: insight into the occasional fertility of mules. Chromosome Res., 12, (2004), 65-76 ZEILMANN, M.: HYPP – hypercalemic periodic paralysis in horses. Tierärztl. Prax., 21, (1993), 524-527

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Chapter 3

Assignment of the COMP gene to equine chromosome 21q12-q14 by FISH and confirmation by RH mapping

D. Müller*, H. Kuiper*, S. Mömke*, C. Böneker*, C. Drögemüller*, B.P. Chowdhary† and O. Distl*

*Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Germany. †Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA

Accepted for publication 17 March 2005

Published in: Animal Genetics 36 (2005) 277-279

Assignment of the gene COMP gene to equine chromosome 21q12-q14 by FISH and confirmation by RH mapping

Assignment of the COMP gene to equine chromosome 21q12-q14 by FISH and confirmation by RH mapping

Source/description: The cartilage oligomeric matrix protein gene (COMP) maps to HSA19p13.1 starting at 18,754,584 bp and ending at 18,763,114 bp and encodes a 524-kD protein that is expressed at high levels in the territorial matrix of chondrocytes1. The human gene consists of 19 exons spanning 8531 bp and the exons 1-3 are unique to COMP, whereas the exons 4-19 encode the EGF-like (type II) repeats, calmodulin-like (type III) repeats (CLRs), and the C-terminal domain correspond in sequence and intron location to the thrombospondin genes2. Mutations in the COMP gene are responsible for both the autosomal dominantly inherited pseudoachondroplasia (PSACH) and some types of multiple epiphyseal dysplasia (MED), which are characterized by mild to severe short-limb dwarfism and early- onset osteoarthritis in man2, 3, 4, 5. Further studies including COMP-null mice revealed that the phenotype in PSACH and MED is caused not by the reduced amount of COMP but by some other mechanism, such as folding defects or extracellular assembly abnormalities due to dysfunctional mutated COMP 6. The equine BAC library CHORI-241 was screened as per standard protocols (http://bacpac.chori.org) with a heterologous 32P-labelled insert of a human COMP cDNA clone (IMAGp998C026158) provided by the Resource Center/Primary Database of the German Human Genome Project (http://www.rzpd.de/). A BAC clone designated CH241-49F5 with an insert of approximately 160 kb was identified. Following culture, BAC DNA was isolated using the Qiagen plasmid midi kit (Qiagen, Hilden, Germany), and end-sequences of the BAC were obtained using the ThermoSequenase kit (AmershamBiosciences, Freiburg, Germany) and a LI-COR 4200 automated sequencer (LI-COR, Inc., Lincoln, Nebraska, USA). The end sequences are deposited in the EMBL nucleotide database (Accession nos. AJ870433 and AJ870434). A BLASTN sequence comparison of the equine SP6 BAC end sequence with the build 35.1 of the human genome sequence revealed a significant match (BLAST E-value 2.6e-38) over 74 bp (93.2% identity) starting at 18,765,397 bp of HSA19p, approximately 11 kb downstream of human COMP.

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Comparison of the T7 end sequence revealed a significant match (BLAST E-value 4.6e-72) over 143 bp (91.6 % identity) starting at 18,529,627 bp of HSA19p, approximately 225 kb upstream of human COMP. The identity of the BAC clone with respect to the presence of the COMP gene was further verified by obtaining primers from an equine EST (Accession no. CX605670) corresponding to exons 14-18 of the human gene, and using them for PCR amplification of the BAC DNA. Sequence of the amplification product when compared with the human genome sequence (build 35.1) revealed a significant match (BLAST E-value 1e-36) over 124 bp (92 % identity) starting at 18,756,781 within exon 16 of the human COMP gene. Primer sequences: Primers for PCR amplification of the 214 bp BAC end sequence (Accession no. AJ870434) and 166 bp internal BAC sequence corresponding to the human COMP gene (Accession no. CX605670) were designed using the PRIMER3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). T7 BAC end sequence: F: 5’-CAC GTC TAG GCA CCT CTC CT-3’ R: 5’-GGC TTG CAT ATC CCC TCA C-3’ Internal BAC sequence: F: 5’-GTT ACA CGG CCT TCA ATG G-3’ R: 5’-CCA CAT GAC CAC GTA GAA GC-3’ Chromosomal location: The equine BAC clone was labelled with digoxygenin using a nick-translation mix (Roche Diagnostics, Mannheim, Germany). FISH on GTG- banded horse chromosomes was performed using 500 ng of labelled DNA, and 20µg sheared equine genomic DNA and 10 µg salmon sperm DNA as competitors. After overnight hybridization, signal detection was performed using a Digoxygenin-FITC Detection Kit (Qbiogene, Heidelberg, Germany). The chromosomes were counterstained with DAPI (4’, 6’-diaminidino-2-phenylindole) and propidium iodide and embedded in antifade. Previously photographed metaphase spreads were re- examined for hybridization signals using a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) equipped for fluorescence. Identification of chromosomes followed the international system for chromosome nomenclature of domestic horses (ISCNH

- 56 - Assignment of the gene COMP gene to equine chromosome 21q12-q14 by FISH and confirmation by RH mapping

1997)7. The equine COMP gene was subsequently mapped to ECA21q12-q14 following examination of 35 metaphase spreads (Fig.1). Radiation hybrid (RH) mapping/PCR conditions: The 5,000-rad TAMU equine radiation hybrid panel8 was genotyped to map the T7 BAC end marker located about 227 kb upstream of COMP. PCR was carried out in a 20 µl volume containing 25 ng of RH cell line DNA, 10 pmol of each primer and 0.85 U Taq polymerase (Qbiogene, Heidelberg, Germany). The reaction conditions were as follows: denaturation at 94°C for 5 min followed by 34 cycles of denaturation for 45 s at 94°C, annealing for 45 s at 62°C and extension for 5 min at 72°C. The PCR was completed with a final cooling at 4°C for 5 min. The amplified products were separated on a 1.5% agarose gel. After scoring the signals, a two-point analysis9 was performed using RHMAPPER-1.22 (http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) against 861 equine markers mapped previously on the first generation whole genome equine RH map. This sequence tagged site (STS) marker showed a retention frequency of 13% and showed close linkage to SG16 (11.99 cR; LOD >12.0) that is located on ECA21q13 by FISH10 and at 38 cR of ECA218 RH map. Thus the FISH and RH results corroborate each other. Comment: The physical assignment of the equine COMP gene on ECA21q12-q14 is in agreement with comparative painting11 but does not agree with comparative mapping of the current equine-human comparative map of ECA21 RH map, which showed conserved synteny to HSA58,12.

Acknowledgements: This study was supported by grants of the German Research Council, DFG, Bonn (DI 333/12-1).

References 1 Newton G. et al. (1994) Genomics 24, 435-9. 2 Briggs M.D. et al. (1995) Nature Genet 10, 330-6. 3 Briggs M.D. et al. (1998) Am J Hum Genet 62, 311-9. 4 Deere M. et al. (1998) Am J Med Genet 80, 510-3. 5 Mabuchi A. et al. (2003) Hum Genet 112, 84-90. 6 Svensson L. et al. (2002) Mol Cell Biol 22, 4366-71.

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7 Bowling A.T. et al. (1997) Chromosome Res 5, 433-43. 8 Chowdhary B.P. et al. (2003) Genome Res 13, 742-51. 9 Slonim D. et al. (1997) J Comput Biol 4, 487-504. 10 Godard S. et al. (1997) Mamm Genome 8, 745-50. 11 Yang F. et al. (2004) Chromosome Res 12, 65-76. 12 Milenkovic D. et al. (2002) Mamm Genome 13, 524-34.

Figure 1 Chromosomal assignment of the equine BAC CH241-49F5 containing COMP by FISH analysis. G-banded metaphase spread before (left) and after hybridization (right). Double signals indicated by arrows are visible on both ECA21 chromosomes.

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Chapter 4

Physical mapping of the PTHR1 gene to equine chromosome 16q21.2

D. Müller*, H. Kuiper*, C. Böneker*, S. Mömke*, C. Drögemüller*, B.P. Chowdhary† and O. Distl*

*Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Germany. †Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA

Accepted for publication 22 March 2005

Published in: Animal Genetics 36 (2005) 282-284

Physical mapping of the PTHR1 gene to equine chromosome 16q21.2

Physical mapping of the PTHR1 gene to equine chromosome 16q21.2

Source/description: The parathyroid hormone receptor 1 (PTHR1) maps to HSA3p22-p21.1 starting at 46,894,240 bp and encodes a receptor for both parathyroid hormone and parathyroid hormone-related protein (PTHRP) that are expressed in most tissues1. Three promoters, P1, P2, and P3, regulate the expression of PTHR1 but the P3 promoter seems to be the most active in human adult kidney and also active in human osteoblast-like cells2. The human gene consists of 16 exons spanning 26054 bp3. The protein encoded by this gene is a member of the G-protein coupled receptor family 24. The activity of this receptor is mediated by G-proteins which activate adenylyl cyclase and also a phosphatidylinositol-calcium second messenger system. Defects in this receptor are known in human to be the cause of Jansen's metaphyseal chondrodysplasia (JMC) and chondrodysplasia Blomstrand type (BOCD), as well as enchondromatosis. Investigations in mouse models suggested that PTHRP mediates chondrocyte differentiation during endochondral ossification. The equine BAC library CHORI-241 was screened to isolate a genomic BAC clone containing the PTHR1 gene. High density BAC colony filters were probed according to the CHORI protocols (http://bacpac.chori.org) with a heterologous 32P-labelled insert of a murine PTHR1 cDNA clone (IRAKp961D1425) provided by the Resource Center/Primary Database of the German Human Genome Project (http://www.rzpd.de/). An equine genomic BAC clone designated CH241-376I8 with an insert of approximately 245 kb containing the PTHR1 gene was identified. BAC DNA was prepared from the BAC clone using the Qiagen plasmid midi kit (Qiagen, Hilden, Germany). BAC DNA sequences were obtained using the ThermoSequenase kit (AmershamBiosciences, Freiburg, Germany) and a LI-COR 4200 automated sequencer (LI-COR, Inc., Lincoln, Nebrasca, USA). The two CH241-376I8 end sequences were deposited in the EMBL nucleotide database under accession numbers AJ870493 and AJ870432, respectively. A BLASTN sequence comparison of the equine SP6 BAC end sequence with the build 35.1 of the human genome

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sequence revealed a significant match (BLAST E-value 5.8e-47) over 286 bp (identity = 83.6%) starting at 47,110,626 bp of HSA3p22, which is approximately 190 kb downstream of human PTHR1. In order to verify the location of the PTHR1 gene on the equine CH241-376I8 BAC, equine expressed sequence tagged (EST) sequences were identified by performing a BLASTN search for est_others of mammalia by using the human mRNA sequence of the PTHR1 gene. An equine EST (Accession no. CX605315) gave a significant hit over 716 bp for exons 11-16 of the human PTHR1 gene (identity = 91 %, BLAST E- value 0.0, Score 967). The equine EST sequence of exon 14 of the human PTHR1 gene was used to derive primers for PCR to obtain equine sequence of PTHR1 from the CH241-376I8 BAC clone. A comparison of this amplification product with the build 35.1 of the human genome sequence revealed a significant match (BLAST E- value 2e-17) over 120 bp (identity = 88 %) starting at 46,919,031 within exon 14 of the human PTHR1 gene. Primer sequences: Primers for PCR amplification of a 212 bp fragment from the CH241-376I8 T7 BAC end sequence (Accession no. AJ870432) and 120 bp internal BAC sequence corresponding to the human PTHR1 gene (Accession no. CX605315) were designed using the PRIMER3 software (http://frodo.wi.mit.edu/cgi- bin/primer3/primer3_www.cgi). T7 BAC end sequence: F: 5’-GGT AGG GGA TGT TAT GGA GAG-3’ R: 5’-ATC ACC TGT CCT GGA AGA GC-3’ Internal BAC sequence corresponding to the human PTHR1 gene: F: 5’-TCA AAT CCA CAC TGG TGC TC-3’ R: 5’-CGT AGT GCA TCT GGA CTT GC-3’ Chromosomal location: The equine BAC clone was labelled with digoxygenin by nick translation using a nick-translation mix (Roche Diagnostics, Mannheim, Germany). FISH on GTG-banded horse chromosomes was performed using 500 ng of digoxygenin labelled BAC DNA, and 20 µg sheared total equine DNA and 20 µg salmon sperm DNA as competitors. After hybridization overnight, signal detection was performed using a Digoxygenin-FITC Detection Kit (Qbiogene, Heidelberg,

- 62 - Physical mapping of the PTHR1 gene to equine chromosome 16q21.2

Germany). The chromosomes were counterstained with DAPI (4’,6’-diaminidino-2- phenylindole) and propidium iodide and embedded in antifade. Previously photographed metaphase spreads were re-examined for hybridization signals using a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) equipped for fluorescence. Identification of chromosomes followed the international system for chromosome nomenclature of domestic horses (ISCNH 1997)5. The equine genomic BAC clone CH241-376I8 containing the PTHR1 gene was located to ECA16q21.2 by examination of metaphase chromosomes of 35 cells (Fig.1). Radiation hybrid (RH) mapping/PCR conditions: To confirm the cytogenetic assignment the 5,000-rad TAMU equine radiation hybrid panel6 was used to map the T7 BAC end located about 192 kb downstream of PTHR1. PCR reaction using the previously described primers for the T7 BAC end was carried out in a 20 µl reaction containing 25 ng of RH cell line DNA, 10 pmol of each primer and 0.85 U Taq polymerase (Qbiogene, Heidelberg, Germany). The reaction conditions started with a denaturing step at 94°C for 5 min followed by 34 cycles using the following protocol: denaturation for 45 s at 94°C, annealing for 45 s at 62°C and extension for 5 min at 72°C. The PCR was completed with a final cooling at 4°C for 5 min. PCR products were separated on a 1.5% agarose gel. After scoring positive signals, a two-point analysis7 was performed using RHMAPPER-1.22 (http://equine.cvm.tamu.edu/cgi- bin/ecarhmapper.cgi) against 861 equine markers mapped previously on the first generation whole genome equine 5,000-rad RH panel6. This sequence tagged site (STS) marker showed a retention frequency of 17.4% and the RH mapping revealed close linkage to PDCD6IP (programmed cell death 6-interacting gene, Accession no. ECA001546) (26.79 cR; LOD >3.0). This gene had been previously mapped on ECA16q21.3 by FISH and by RH mapping on ECA16 at 112.5 cR8. On the human gene map, PDCD6IP is located at HSA3p22.3 starting at 33,815,086 bp and ending at 33,883,502 bp (human genome map viewer build 35.1). Thus, the RH results confirm the result obtained by FISH. Comment: The physical assignment of the equine PTHR1 gene on ECA16q21.2 agrees with comparative mapping of the current equine-human comparative RH6 and

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cytogenetic map8 at the chromosomal position of ECA16q21. These results confirm the conserved synteny of ECA16 to a small segment of HSA3p represented by cytogenetically mapped loci located on HSA3p21 (APEH, acylamino acidreleasing enzyme), HSA3p21.3 (GPX1, glutathione peroxidase 1), HSA3p22 (TGFBR2, transforming growth factor beta, type II), and HSA3p25 (HRH1, histamine receptor H1)8.

Acknowledgements: This study was supported by grants of the German Research Council, DFG, Bonn (DI 333/12-1).

References 1 Pausova Z. et al. (1994) Genomics 20, 20-6. 2 Manen D. et al. (2000) J Clin Endocr Metab 85, 3376-82. 3 McCuaig K.A. et al. (1994) Proc Nat Acad Sci 91, 5051-5. 4 Juppner H. et al. (1991) Science 254, 1024-6. 5 Bowling A.T. et al. (1997) Chromosome Res 5, 433-43. 6 Chowdhary B.P. et al. (2003) Genome Res 13, 742-51. 7 Slonim D. et al. (1997) J Comput Biol 4, 487-504. 8 Milenkovic D. et al. (2002) Mamm Genome 13, 524-34.

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Figure 1 Chromosomal assignment of the equine BAC CH241-376I8 containing PTHR1 by FISH analysis. G-banded metaphase spread before (left) and after hybridization (right). Double signals indicated by arrows are visible on both ECA16 chromosomes.

- 65 -

Chapter 5

Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5 and SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping

D. Müller*, H. Kuiper*, C. Böneker*, S. Mömke*, C. Drögemüller*, B.P. Chowdhary† and O. Distl*

*Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Hannover, Germany. †Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA

Accepted for publication 12 June 2005

Published in: Animal Genetics 36 (2005) 457-460

Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5 and SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping

Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5, and SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping

Source/description: The human bone gamma-carboxyglutamate (gla) protein (osteocalcin) gene (BGLAP) consists of four exons spanning 1,108 bp1. BGLAP encodes a 100 amino acid protein that is expressed in osteoblasts but not in other extracellular matrix producing cell types2. Polymorphisms of the osteocalcin gene are associated with bone mineral density in postmenopausal women3. Through testing for linkage and association simultaneously the osteocalcin gene was identified as a quantitative trait locus (QTL) underlying hip bone mineral density variation4. The bone morphogenetic protein 2 gene (BMP2) belongs to the transforming growth factor-beta (TGFB) superfamily. The human gene consists of three exons spanning 10,563 bp5. BMP2 is regarded as a multifunctional cytokine involved in inducing the formation and regeneration of cartilage and bone6, and it also participates in organogenesis, cell differentiation, cell proliferation, and apoptosis. BMP2 has an essential role in the regulation of the steps of chondrocyte and osteoblast differentiation and it induces osteocalcin expression in osteoblasts7. The BMP2 gene has been suggested as a candidate for fibrodysplasia (myositis) ossificans progressiva in man, which is characterized by intermittently progressive ectopic ossification and malformed big toes which are often monophalangic8. The human carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 4 (CHST4) gene encodes a protein with 386 amino acids that is involved in in vitro enzymatic synthesis of the disulfated disaccharide unit of corneal keratan sulfate9,10. The human gene consists of two exons spanning 12,358 bp. The solute carrier family 1 (glial high affinity glutamate transporter), member 3 or excitatory amino acid transporter 1 gene (SLC1A3, EAAT1) belongs to the family of high-affinity sodium-dependent transporter molecules that regulates neurotransmitter concentrations at the excitatory glutamatergic synapses of the mammalian central nervous system11,12. An important function of the SLC1A3 protein is the re-uptake of

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secreted amino acid neurotransmitter, possibly maintaining extracellular amino acid concentrations at nontoxic and nonepileptogenic levels11,12. The human SLC1A3 gene consists of 10 exons spanning 81,750 bp13 and encodes a 542-amino acid glutamate transporter protein11,12. SLC1A3 appears to be related to the syndrome of microcephaly and mental retardation in association with an interstitial deletion of the distal chromosomal band of HSA5p1314. Aberrant glutamate transporter expression is involved in Alzheimer's disease as a mechanism of neurodegeneration as shown by expression patterns in a subset of cortical pyramidal neurones in dementia cases with Alzheimer-type pathology15. The solute carrier family 4, anion exchanger, member 1 (erythrocyte membrane protein band 3, Diego blood group, band 3 of red cell membrane) gene (SLC4A1, BND3) is the major glycoprotein of the erythrocyte membrane where it mediates exchange of chloride and bicarbonate across the phospholipid bilayer and plays a central role in carbon dioxide transport from tissues to lungs16,17. Many SLC4A1 mutations are known in man and these mutations can lead to two types of diseases; destabilization of red cell membrane leading to hereditary spherocytosis and defective kidney acid secretion leading to distal renal tubular acidosis18,19,20. Other SLC4A1 mutations that do not give rise to disease result in novel blood group antigens, which form the Diego blood group system. Southeast Asian ovalocytosis (SAO, Melanesian ovalocytosis) results from the heterozygous presence of a deletion in the SLC4A1 protein and is common in areas where Plasmodium falciparum malaria is endemic21. The human SLC4A1 gene consists of 20 exons spanning 18,428 bp1. The solute carrier family 9 (sodium/hydrogen exchanger), isoform 5 gene (SLC9A5, NHE5) is the fifth member of plasma membrane proteins that mediate the exchange of extracellular Na+ for intracellular H+1. Mammalian Na+/H+ exchangers (NHEs) are a family of integral membrane proteins that play a central role in sodium, acid-base, and cell volume homeostasis. Its function is dynamically regulated by phosphatidylinositol 3'-kinase and by the state of F-actin assembly22. The human SLC9A5 gene consists of 16 exons spanning 23,231 bp1 and encodes an 896-amino acid plasma membrane protein22.

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The solute carrier family 20 (phosphate transporter), member 1 gene (SLC20A1 or GLVR1, gibbon ape leukaemia virus receptor 1) is a sodium-dependent phosphate symporter which acts as a retrovirus receptor allowing infection of human and murine cells for gibbon ape leukaemia virus23. The human SLC20A1 gene consists of 11 exons spanning 17,876 bp24 and encodes a 679-amino acid membrane protein containing multiple transmembrane domains25. BAC library screening/sequence analysis: The equine CHORI-241 BAC library was screened for BAC clones containing the selected genes. High density BAC colony filters were probed according to CHORI protocols (http://bacpac.chori.org) with heterologous 32P-labelled inserts of the respective human or murine cDNA IMAGE clones provided by the Resource Center/Primary Database of the German Human Genome Project (http://www.rzpd.de/). BAC DNA was prepared from positive BAC clones using the Qiagen plasmid midi kit (Qiagen, Hilden, Germany). BAC DNA end sequences were obtained using the ThermoSequenase kit (AmershamBiosciences, Freiburg, Germany) and a LI-COR 4200 automated sequencer (LI-COR Inc., Lincoln, NE, USA). BAC subclone sequences were obtained for the BAC DNA containing the SLCA1A3 gene because BLASTN sequence comparisons of SP6 and T7 termini of this BAC clone gave no significant hits to human genome sequences (Build 35.1). For this clone, BAC DNA was digested with EcoR I (New England Biolabs, Schwalbach, Germany) and separated on 0.8% agarose gels in order to obtain internal BAC DNA sequences. The resulting fragments were cloned into the polylinker of pGEM-4Z (Promega, Mannheim, Germany) and then transformed into XL1-Blue competent Escherichia coli. Montage Plasmid Miniprep kit (Millipore, Molsheim, France) was used to isolate 16 subclones for sequencing. BLASTN sequence comparisons of the equine BAC sequences were performed against Build 35.1 of the human genome sequence. In order to verify the selected genes on the equine BAC clones, sequence primers were derived using human exonic sequences of these genes. Amplification products of these internal BAC sequences were verified by BLASTN sequence comparisons with the Build 35.1 of the human genome sequence. Primers for PCR amplification from the BAC sequences were designed using the PRIMER3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).

- 71 - Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5 and SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping

The results of the BLASTN sequence comparisons of the equine BAC end and internal sequences with the Build 35.1 of the human genome sequences are shown in Table 1. Chromosomal location: The equine BAC clones were labelled with digoxygenin by nick translation using a nick-translation mix (Roche Diagnostics, Mannheim, Germany). FISH on GTG-banded horse chromosomes was performed using 500 ng digoxygenin labelled BAC DNA, and 20 µg sheared total equine DNA and 10 µg salmon sperm DNA as competitors. After hybridization overnight, signal detection was performed using a digoxygenin-FITC detection kit (Qbiogene, Heidelberg, Germany). The chromosomes were counterstained with DAPI (4’,6’-diaminidino-2- phenylindole) and propidium iodide and embedded in antifade. Previously photographed metaphase spreads were re-examined for hybridization signals using a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) equipped for fluorescence. Identification of chromosomes followed the international system for chromosome nomenclature of domestic horses (ISCNH 1997)26. The equine genomic BAC clones containing the respective genes were located to equine chromosomes by examination of at least 30 metaphase spreads (Fig. 1a-g). Radiation hybrid mapping: The 5,000-rad TAMU equine radiation hybrid (RH) panel27 was genotyped to map the SP6 or T7 BAC end markers. PCR was carried out in a 20 µl reaction containing 25 ng RH cell line DNA, 15 pmol of each primer and 0.75 U Taq polymerase (Qbiogene, Heidelberg, Germany). The reaction conditions started with a denaturing step at 94°C for 5 min followed by 34 cycles using the following protocol: denaturation for 45 s at 94°C, annealing for 45 s at 60°C and extension for 5 min at 72°C. The PCR was completed with a final cooling at 4 °C for 5 min. PCR products were separated on a 1.5% agarose gel. After scoring positive signals, a two-point analysis28 was performed using RHMAPPER-1.22 (http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) against 861 equine markers mapped previously on the first generation whole genome equine RH map. The FISH and RH mapping results and the conserved synteny to human chromosomes are given in Table 2. The lod scores of all closest linked markers were > 3.0. The RH results confirmed the results obtained by FISH.

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Comment: The physical assignments of these seven equine genes agreed with the current equine-human comparative RH map27,29.

Acknowledgement: This study was supported by grants of the German Research Council, DFG, Bonn (DI 333/12-1).

References 1 International Human Genome Sequencing Consortium (2004) Nature 431, 931-45. 2 Kerner S. A. et al. (1989) Proc Natl Acad Sci USA 86, 4455-9. 3 Yamada Y. et al. (2003) J Clin Endocrinol Metab 88, 3372-8. 4 Deng H. W. et al. (2002) J Bone Miner Res 17, 678-86. 5 Deloukas P. et al. (2001) Nature 414, 865-71. 6 Majumdar M. K. et al. (2001) J Cell Physiol 189, 275-84. 7 Valcourt U. et al. (2002) J Biol Chem 277, 33545-58. 8 Connor J., Evans D.A.P. (1982) J Bone Joint Surg 64, 76-83. 9 Akama T.O. et al. (2002) J Biol Chem 277, 42505-13. 10 Martin J. et al. (2004) Nature 432, 988-94. 11 Arriza J. L. et al. (1994) J Neurosci 14, 5559-69. 12 Stoffel W. et al. (1996) FEBS Lett 386,189-93. 13 Schmutz J. et al. (2004) Nature 431, 268-74. 14 Keppen L. D. et al. (1992) Am J Med Genet 44, 356-60. 15 Scott H. L. et al. (2002) J Neurosci 22, RC206. 16 Palumbo A. P. et al. (1986) Am J Hum Genet 39, 307-16. 17 Langdon R. G., Holman V.P. (1988) Biochim Biophys Acta 945, 23-32. 18 Tanner M.J. (2002) Curr Opin Hematol 9,133-9. 19 Sritippayawan S. et al. (2004) Am J Kidney Dis 44, 64-70. 20 Shayakul C. et al. (2004) Nephrol Dial Transplant 19, 371-9. 21 Allen S. J. et al. (1999) Am J Trop Med Hyg 60, 1056-60. 22 Szaszi K. et al. (2002) J Biol Chem 277, 42623-32. 23 Kavanaugh M.P. et al. (1994) Proc Natl Acad Sci 91, 7071-5. 24 Palmer G. et al. (1999) Gene 226, 25-33.

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25 O'Hara B. et al. (1990) Cell Growth Diff 1, 119-27. 26 Bowling A. T. et al. (1997) Chromosome Res 5, 433-43. 27 Chowdhary B. P. et al. (2003) Genome Res 13, 742-51. 28 Slonim D. et al. (1997) J Comput Biol 4, 487-504. 29 Milenkovic D. et al. (2002) Mamm Genome 13, 524-34.

Figure 1 Chromosomal assignment of the equine BACs containing a) BGLAP, b) BMP2, c) CHST4, d) SLC1A3, e) SLC4A1, f) SLC9A5 and g) SLC20A5 by FISH analysis. G-banded metaphase spread before (left) and after (right) hybridization. Double signals indicated by arrows are visible on both equine chromosomes. (a)

(b)

- 74 - Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5 and SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping

(c)

(d)

(e)

- 75 - Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5 and SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping

(f)

(g)

- 76 - Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5 and SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping

Table 1 Selected genes, their human location and cDNA clone identity, insert size of the positive equine BAC clones, used BAC end sequence, Accession no., BLASTN sequence comparisons of equine BAC sequences with human genome and their locations on human genome (Build 35.1). Gene HSAa Human Human or murine BAC BAC end Accession or no. location cDNA clone identity insert internal (bp) size (IN) (kb) sequence (Build 35.1) BGLAP

153,025, CHORI- SP6 AJ937344 078 241- 1q25- 153,026, 256K13 T7 AJ937345 q31 185 IMAGp998K204334b 195 IN AJ937346 BMP2 CHORI- 241- 6,697,207 159E8 T7 AJ871594 20p12 6,707,769 IMAGp998G015200b 230 IN CX593098 CHST4

70,117, CHORI- SP6 AJ937339 560 241- 70,129, 84A13 T7 AJ937340 16q22.2 917 IMAGp998I158096b 221 IN AJ937341 SLC1A3

36,642, CHORI- d 446 241- SC AJ937342 36,724, 406K6 5p13 195 IRALp962P1337c 200 IN AJ937343 SLC4A1 39,682, CHORI- 566 241- 17q21- 39,700, 313L9 T7 AJ937334 q22 993 IMAGp998C13535c 190 IN AJ937333

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Table 1 continued Gene HSAa Human Human or murine BAC BAC end Accession or no. location cDNA clone identity insert internal (bp) size (IN) (kb) sequence (Build 35.1) SLC9A5

65,840, CHORI- SP6 AJ937330 365 241- T7 AJ937331 65,863, 141P15 16q22.1 595 IMAGp998M093449c 145 IN AJ937332 SLC20A1 113,119, CHORI- 758 241- 2q11- 113,137, 172C18 SP6 AJ937336 q14 633 IRAKp961B1026b 170 IN AJ937338

Table 1 continued Gene BLAST Length of Identity Beginning of Distance Match to E-Value match to human (bp) (%) sequence selected gene alignment gene (Build on HSA (bp) (kb) 35.1)

BGLAP exon 12 1.4e-55 217 88% 153,247,625 222 of MEF2D

41 92% 152,988,960 36 -

2.0e-5 58 87% 152,988,870 36 - exon 3 of 1.0e-4 46 89% 153,025,659 0 BGLAP BMP2 1.1e-134 385 85% 6,807,012 99 - exon 3 of 0.0 578 90% 6,706,890 0 BMP2

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Table 1 continued Gene BLAST Length of Identity Beginning Distance Match to E-Value match of to human (bp) (%) sequence selected gene alignment gene (Build on HSA (kb) 35.1) (bp) CHST4 2.0e-19 112 87% 69,910,086 208 -

4.0e-3 73 84% 70,131,053 1,1 -

4.0e-6 122 81% 70,131,184 1,1 - exon 2 of 5.0e-129 624 84% 70,128,532 0 CHST4 exon 2 of 4.0e-37 130 90% 70,128,362 0 CHST4 SLC1A3 3.0e-11 236 86% 36,786,831 63 -

2.0e-61 408 83% 36,786,935 63 - exon 4 and intron 4 of 1.0e-111 443 84% 36,706,960 0 SLC1A3

4.0e-13 110 80% 36,707,388 0 - SLC4A1 3.7e-41 120 87% 39,680,601 2 - exon 10 of 4.0e-53 288 82% 39,690,640 0 SLC4A1 SLC9A5 exon 2 of 4.0e-179 457 92% 65,911,858 48 FLJ40162 intron 8 of 3.0e-15 57 96% 65,729,633 111 Lin10 exon 16 of 3.0e-59 251 82% 65,862,389 0 SLC9A5 exon 16 of 3.0e-59 171 79% 65,862,807 0 SLC9A5

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Table 1 continued Gene BLAST Length of Identity Beginning of Distance Match to E-Value match to human (bp) (%) sequence selected gene alignment gene (Build on HSA (bp) (kb) 35.1)

SLC20A1 5.5e-61 190 91% 113,182,799 45 - exon 8 of 2.0e-88 252 91% 113,133,335 0 SLC20A1 a: HSA- homo sapiens autosome b: human cDNA clone c: murine cDNA clone

Table 2 Selected genes, equine BAC ends and PCR primer sequences used for radiation hybrid (RH) mapping on the 5,000-rad TAMU equine RH panel27, equine genome location by FISH and RH mapping, and conserved synteny to the human genome (Build 35.1). Gene BAC enda Primer Sequences (5`- 3`) PCR

(bp) F: ATG GGT GGA TCT GGG TAC TG BGLAP SP6 R: GGA GGA AGA CTG GCA CAG AT 175 F: TTT GGA CTG CTT AAA CCA TTG BMP2 T7 R: CTT GCC CAA AGG ACC TCT C 226 F: CTG TTG TCT GCC ACT AAG AAT G CHST4 SP6 R: TGT GTG CCA TTA GTG GAA TG 751 F: AAC AAG CCT TCAT CAT CAG C SLC1A3 SCb R: ATT GGC TTT GTG TTT GCT TC 197 F: TTT TGC CTT AGT TCC GTG TC SLC4A1 T7 R: ATA TGG TAC TTG GGC CTT GC 188

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Table 2 continued Gene BAC enda Primer Sequences (5`- 3`) PCR

(bp) F: GCC AGA GGC TAT TCA TCG AC SLC9A5 T7 R: AAG AGG GTC ACC TCA GCA AC 249 F: ACA ATA GCT GCC TGG CAT AG SLC20A1 T7 R: TTC TGC CTG CAT TTA GCT TC 222

Table 2 continued Gene RH-mapping Location Conserved on horse synteny to genome Closest marker Distance (cR)f RET (%)g ECAd HSAe

BGLAP VHL66 51.42 20.70% 5p12-p13 1q21-q32

BMP2 HTG14 17.91 19.60% 22q14 20p12-p13

CHST4 AHT022 4.5 cR 21.70% 3p13 16q

SLC1A3 IL7R 0.0 19.60% 21q17 5p12-p14 11p12.1- SLC4A1 SG24 5.2 cR 26.10% p12.3 17q21-q25

SLC9A5 CBFB 0.10 22.80% 3p13 16q12-q24

SLC20A1 IL1B 0.0 19.60% 15q13 2q13-q21

- 81 -

Chapter 6

Physical mapping of the ATP2A2 gene to equine chromosome 8p14->p12 by FISH and confirmation by linkage and RH mapping

D. Müller*, H. Kuiper*, C. Böneker*, C. Drögemüller, * J.E. Swinburne, M. Binns, B.P. Chowdhary and O. Distl*

*Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Hannover, Germany. †Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA

Accepted for publication 16 December 2005

Published in: Cytogenetic and Genome Research 114 (2006) 94

Physical mapping of the ATP2A2 gene to equine chromosome 8p12-p14 by FISH and confirmation by linkage and RH mapping

Physical mapping of the ATP2A2 gene to equine chromosome 8p12-p14 by FISH and confirmation by linkage and RH mapping

Rationale and significance The ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 gene (ATP2A2) encodes one of the SERCA Ca(2+)-ATPases, which are intracellular pumps located in the sarcoplasmic or endoplasmic reticula of muscle cells1,2. This enzyme catalyzes the hydrolysis of ATP coupled with the translocation of calcium from the cytosol to the sarcoplasmic reticulum lumen, and is involved in regulation of the contraction/relaxation cycle1,2. ATP2A2 maps to HSA12q23-q24.13 at 109,182,152 to 109,251,615 and consists of 21 exons spanning 69464 bp4. Mutations in the ATP2A2 gene cause the autosomal dominantly inherited keratosis follicularis, also known as Darier-White disease, characterized by loss of adhesion between epidermal cells and abnormal keratinization5,6,7. Further studies including a null mutation in one copy of the ATP2A2 gene in mice revealed that serca2 protein levels were reduced by about a third, sarcoplasmic reticulum calcium stores decreased and in isolated cardiomyocytes released. In addition, these mice had reduced myocyte contractility8. Aged heterozygous mutant (Atp2a2 +/-) mice developed squamous cell tumors, which led to the conclusion that Atp2a2-haploinsufficiency predisposes murine keratinocytes to neoplasia, and that perturbation of Ca(2+) homeostasis or signaling can be a primary initiating event in cancer9.

Materials and methods BAC isolation and characterization of the equine ATP2A2 clone The equine BAC library CHORI-241 was screened for a BAC clone containing the ATP2A2 gene. High density BAC colony filters were probed according to CHORI protocols (http://bacpac.chori.org) with a heterologous 32P-labelled insert of a human ATP2A2 cDNA clone (IMAGp998K2010072) from the Resource Center/Primary

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Database of the German Human Genome Project (http://www.rzpd.de/). An equine genomic BAC clone with an insert of approximately 220 kb containing the ATP2A2 gene was identified and designated CH241-167G5. BAC DNA was prepared from the BAC clone using the Qiagen plasmid midi kit (Qiagen, Hilden, Germany). BAC DNA end sequences were obtained using the ThermoSequenase kit (AmershamBiosciences, Freiburg, Germany) and a LI-COR 4200 automated sequencer (LI-COR Inc., Lincoln, NE, USA). The CH241-167G5 SP6 and T7 end sequences were deposited in the EMBL nucleotide database under accession nos. AJ937347 and AJ937348. A BLASTN sequence comparison of the equine SP6 BAC end sequence with the Build 35.1 of the human genome sequence revealed four significant matches (BLAST E-value of each match 1.4e-65). The first match was over 101 bp (identity = 89.1 %), which started at 109,369,448 bp of HSA12q24.11 approximately 118 kb downstream of human ATP2A2. The further BLASTN matches over 63, 61, and 30 bp started at 109,369,792 bp, 109,369,269 bp and 109,369,371 bp of HSA12q24.11. Two of the three BLASTN matches were located within exon 1 of the HSU79274 (protein predicted by clone 23733) on HSA12q24.11. The identity of the BAC clone with respect to the presence of the ATP2A2 gene was further verified by obtaining DNA sequences from a shotgun plasmid library generated from the BAC. For this, DNA from the clone CH241-167G5 was isolated using the Qiagen Large Construct kit (Qiagen, Hilden, Germany). BAC DNA was mechanically sheared to obtain fragments of approximately 2kb, and subsequently used to construct a shotgun plasmid library. In total 96 plasmid subclones were sequenced with the ThermoSequenase kit (AmershamBiosciences, Freiburg, Germany) and a LICOR 4200 automated sequencer (LI-COR Inc., Lincoln, NE, USA). Sequence data were analyzed with Sequencher 4.1.4 (GeneCodes, AnnArbor, MI, USA). Sequences of the shotgun plasmid library (accession no. AJ937349) when compared with the human genome sequence (Build 35.1) revealed a significant match (BLAST E-value 1e-64) over 228 bp (87 % identity) starting at 109,223,486 with exon 6 and parts of intron 5 and 6 of the human ATP2A2 gene. A (GT)25 dinucleotide repeat was identified in the sequence was identified in the sequence, and flanking PCR primers ATP2A2_MS5_F: 5`-TAA GAA GAA AGA GAG TGA TG-3`and

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ATP2A2_MS5_R: 5`-GCT TTT CGT TTG GAA TAA ACC-3` were obtained for the microsatellite (AJ937349) using the PRIMER3 software (http://frodo.wi.mit.edu/cgi- bin/primer3/primer3_www.cgi).

PCR conditions and microsatellite analysis The PCR amplification (12 µl final volume) was performed on a PTC 200TM thermal cycler (MJ Research, Watertown, MA, USA) using 20 ng of genomic equine DNA, 1.2 µl 10 x PCR buffer, 0.24 µl DMSO, 0.2 µl dNTPs (5 mM each), 0.5 µl of each primer (10 pmol/µl) and 0.5 Taq DNA polymerase (Qbiogene, Heidelberg, Germany). One primer of the pair was endlabeled with fluorescent IRD 700 to enable flourescent PCR fragment detection. Reaction started with a denaturing step at 94ºC for 4 min followed by 35 cycles using the following protocol: denaturation for 30 s at 94ºC, annealing for 30 s at 58ºC and extension for 45 s at 72ºC. The PCR was completed with a final cooling at 4ºC for 20 min. Raw data were genotyped using Gene Profiler 3.55 software (Scanalytics Inc., Fairfax, USA). Marker characteristics were determined by genotyping horses of three different breeds and the two three- generation, full-sib reference families with 67 offspring created at the Animal Health Trust, Newmarket, UK (NRF)13 using the above mentioned PCR primer pair. (Table 1).

Chromosome preparation Equine metaphase spreads for FISH on GTG-banded chromosomes were prepared using phytohemagglutinin stimulated blood lymphocytes from a normal horse. Cells were harvested and slides prepared using standard cytogenetic techniques. Prior to fluorescence in situ hybridization the chromosomes were GTG-banded and well- banded metaphase chromosomes were photographed using a highly sensitive CCD camera and IPLab 2.2.3 software (Scanalytics).

Fluorescence in situ hybridization The equine BAC clone CH-241-167G5 containing the equine ATP2A2 gene was labelled with digoxygenin by nick translation using a nick-translation mix (Roche

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Diagnostics, Mannheim, Germany). FISH on GTG-banded horse chromosomes was performed using 500 ng of digoxygenin labelled BAC DNA, and 20 µg sheared total equine DNA and 10 µg salmon sperm DNA were used as competitors. After hybridization overnight, signal detection was performed using a digoxygenin-FITC detection kit (Qbiogene, Heidelberg, Germany). The chromosomes were counterstained with DAPI (4’,6’-diaminidino-2-phenylindole) and propidium iodide and embedded in antifade. Previously photographed metaphase spreads were re- examined for hybridization signals using a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) equipped for fluorescence. Chromosomes were identified according to the international system for chromosome nomenclature of domestic horses (ISCNH 1997)10. The equine genomic BAC clone CH241-167G5 containing the ATP2A2 gene was mapped to ECA8p12-p14 following examination of 37 metaphase spreads (Fig. 1). Probe name: CH241-167G5 Probe type: BAC clone from equine genomic BAC library CHORI-241 Insert size: 220 kb Vector: pCMV-Sport6 Proof of authenticity: PCR and DNA sequencing Gene reference for the human ATP2A2 gene: Sakunthabhai et al. (1999)

Radiation hybrid mapping To confirm the cytogenetic assignment the 5,000-rad TAMU equine radiation hybrid (RH) panel11 was genotyped to map the SP6 BAC end marker located about 118 kb downstream of ATP2A2. PCR was carried out in a 20 µl reaction containing 25 ng RH cell line DNA, 10 pmol of each primer and 0.85 U Taq polymerase (Qbiogene, Heidelberg, Germany). The reaction conditions started with a denaturing step at 94°C for 5 min followed by 34 cycles using the following protocol: denaturation for 45 s at 94°C, annealing for 45 s at 60°C and extension for 5 min at 72°C. The PCR was completed with a final cooling at 4 °C for 5 min. PCR products were separated on a 1.5% agarose gel. After scoring positive signals, a two-point analysis12 (http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) was conducted to find

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associations between ATP2A2 versus the markers of the first generation whole genome equine RH map. This sequence tagged site (STS) marker showed a retention frequency of 21.7 % and revealed close linkage to UM034 (22.06 cR; LOD >3.0). The linked microsatellite marker had been previously mapped on ECA8 by RH mapping11. Thus the FISH and RH results corroborate each other. The most closely linked equine genes, SART3 (squamous cell carcinoma antigen recognised by T cells 3) and FLJ11021 (similar to splicing factor, arginine/serine-rich 4), on ECA8 were also mapped on HSA12q24 and HSA12q24.3111.

Comment: The physical assignment of the equine ATP2A2 gene on ECA8p12-p14 agrees with the current equine-human comparative ECA8 RH map, which showed conserved synteny to HSA12q2411.

Linkage mapping The two full-sib reference families with 67 offspring (NRF)13 were genotyped to map the microsatellite marker located within intron 5 of ATP2A2. PCR conditions, primer pairs and fragment analysis were as described above. Linkage analysis was performed using CRI-MAP program version 2.4 (Green et al. 1990). Maximum likelyhood estimates of recombination fraction (theta) were calculated using the TWO-POINT option with a lod-score threshold > 3 to determine linkage group and chromosomal assignment versus the markers of the comprehensive horse linkage map including 626 markers linearly ordered and 140 markers assigned to a chromosomal region14.

Results and discussion Chromosomal location The equine genomic BAC clone CH241-167G5 containing the ATP2A2 gene was mapped to ECA8p12-p14 following examination of 37 metaphases spreads (Fig.1). Mapping data Most precise location: ECA8p12-p14 Number of cells examined: 37

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Number of cells with specific signals: 0(10), 1(4), 2(9), 3(7), 4(7) chromatids per cell Location of background signals (site with > 2 signals): none observed RH analysis of the sequence tagged site (STS) marker of the BAC clone CH241- 167G5 showed a retention frequency of 21.7%. Two-point linkage analysis revealed close linkage to the microsatellite marker was previously mapped on ECA8 at 58.7 cM of the horse linkage map14 and by RH-mapping at 17.3 cR of ECARH08b11. The microsatellite ATP2A2_MS5 showed recombination fractions of 0.06 with ECA microsatellites LEX023 (Lod score = 23.7) at 71.0 cM and UCDEQ46 (Lod score = 17.8) at 58.7 cM. Thus the FISH, RH and genetic linkage results corroborate each other. The most closely linked equine genes, SART3 (Squamous cell carcinoma antigen recognised by T-cells 3) and FLJ11021 (similar to splicing factor, arginine/serine-rich 4), on ECA8 were also mapped on HSA12q24 and HSA12q24.3111. The physical assignment of the equine ATP2A2 gene on ECA8p12-p14 agrees with the current equine-human comparative ECA8 RH map, which showed conserved synteny to HSA12q2411.

Acknowledgements This study was supported by grants of the German Research Council (DFG), Bonn (DI 333/12-1).

References 1 MacLennan D. H. et al. (1985) Nature 316, 696-700. 2 Ruiz-Perez V. L. et al. (1999) Hum Molec Gene. 8, 1621-30. 3 Otsu K. et al. (1993) Genomics 17, 507-9. 4 International Human Genome Sequencing Consortium (2004) Nature 431, 931-45. 5 Sakuntabhai A. et al. (1999) Nat Genet 21, 271-7. 6 Sakuntabhai A. et al. (1999) Hum Molec Genet 8, 1611-9. 7 Jacobsen N. J. O. et al. (1999) Hum Molec Genet 8, 1631-6.

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8 Ji Y. et al. (2000) J Biol Chem 275, 38073-80. 9 Liu L. H. et al. (2001) J Biol Chem 276, 26737-40. 10 Bowling A. T. et al. (1997) Chromosome Res 5, 433-43. 11 Chowdhary B. P. et al. (2003) Genome Res 13, 742-51. 12 Slonim D. et al. (1997) J Comput Biol 4, 487-504. 13 Swinburne et al. (2000) Genomics 66: 123-134 14 Penedo et al. (2005) Cytogenet. Genome Res. 111: 5-15

Figure 1 Chromosomal assignment of the equine BAC CH241-167G5 containing ATP2A2 by FISH analysis. G-banded metaphase spread before (left) and after (right) hybridization. Double signals indicated by arrows are visible on both ECA8 chromosomes.

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Table 1 Characterization of the newly developed ATP2A2-associated microsatellite ATP2A2_MS5 Breed/source Sample size No. of Allele size Expected PICa alleles min-max heterozygozity (bp) South German coldblood 10 8 131-177 0.90 0.76 Saxon- Thuringa coldblood 6 5 131-177 0.83 0.68

Hannoverian warmblood 10 7 131-171 0.90 0.76

NRFb 67 8 135-171 0.87 0.72 aPolymorphism Information Content; Hardy-Weinberg equilibrium in each population genotyped; bNRF:

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Chapter 7

Assignment of the COL8A2 gene to equine chromosome 2p15-p16 by FISH and confirmation by RH mapping

C. Böneker*, D. Müller*, H. Kuiper*, C. Drögemüller*, B.P. Chowdhary+ and O. Distl*

*Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Hannover, Germany. +Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomechanical Sciences, Texas A & M University, College Station, TX 77843, USA.

Accepted for publication 1 May 2005

Published in: Animal Genetics 36 (2005) 444-445

Assignment of the COL8A2 gene to equine chromosome 2p15-p16 by FISH and confirmation by RH-mapping

Assignment of the COL8A2 gene to equine chromosome 2p15-p16 by FISH and confirmation by RH mapping

Source/description: The human collagen, type VIII, alpha 2 (COL8A2) gene encodes the alpha 2 chain of type VIII collagen, which is a major component of the Descemet basement of corneal endothelial cells. The human gene encoding the alpha 2 chain of type VIII collagen consists of two exons spanning about 5,005 bp. The COL8A2 gene has been assigned to HSA1p34.3-p32.3 at 36,229,939 bp to 36,234,943 bp1. A region of 6 to 7 cM on human chromosome 1p34.3-p32, which includes the COL8A2 gene, was linked in a family with early-onset FECD (Fuchs endothelial corneal dystrophy)2. Analysis of the coding sequence of COL8A2 defined a Gln 455 Lys missense mutation in this family2. Analysis in other FECD patients demonstrated further missense substitutions in familial and sporadic cases of FECD, as well as in a single family with posterior polymorphous corneal dystrophy (PPCD)2. The corneal endothelial dystrophies arise from dysfunction of the corneal epithelium leading to corneal opacification2. The underlying pathogenesis of FECD and PPCD may be related to a disturbance of the role of type-VIII collagen in its influence on the terminal differentiation of the neural crest-derived corneal endothelial cell2, so that COL8A2 is a candidate gene for corneal endothelial dystrophies. The equine BAC CHORI-241 library was screened for a BAC clone containing the COL8A2 gene. High-density BAC colony filters were probed according to CHORI protocols (http://www.chori.org/bacpac/) with a heterologous 32P-labelled insert of a human COL8A2 cDNA clone (IMAGp998A122908) from the Primary Database of the Resource Center of the German Human Genome Project (http://www.rzpd.de/). An equine genomic BAC clone with an insert of approximately 170 kb containing the COL8A2 gene was identified and designated CH241-184G10. BAC DNA was prepared from 100 mL overnight cultures using the Qiagen Midi plasmid kit according to the modified protocol for BACs (Qiagen, Hilden, Germany). BAC ends were sequenced using the ThermoSequenase kit (Amersham Biosciences, Freiburg, Germany) and a LI-COR 4300 automated sequencer (LI-COR Inc., Lincoln, NE, USA). The BAC end sequences were deposited in the EMBL nucleotide database

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(accession nos. AJ891045 and AJ891046). BLASTN analysis of the CH241-184G10 SP6 BAC end against Build 35.1 of the human genome revealed a significant match on HSA1p35-p34 located approximately 181 kb upstream of the human COL8A2 gene (identity = 88%, BLAST E-value 1e-41) in the human EIF2C1 gene starting at 36,048,900 bp in exon 14. Furthermore, a BLASTN sequence comparison of an equine BAC sequence derived from an equine expressed sequence tag (EST, accession no. CX605143) corresponding to human COL8A2 gave three significant non-overlapping matches (BLAST E-value 1e-34) located in exon 2 of human COL8A2 gene starting at 36,231,100 bp (Build 35.1). The first match was over 107 bp (identity = 84%), the second match over 111 bp (identity = 84%) and the third match over 165 bp (identity = 86%). Primer sequences: Primers for PCR amplification of a 113 bp product from the CH241-184G10 SP6 BAC end sequence (accession no. AJ891045) and a 590 bp internal BAC sequence matching with exon 2 of human COL8A2 (accession no. CX605143) were designed using the PRIMER3 software (http://frodo.wi.mit.edu/cgi- bin/primer3/primer3_www.cgi). SP6 BAC end sequence: F: 5’-TGA GCC CCC AAG ACT ACA C-3’ R: 5’-TGA AGA TAC CAA GGG AGT GG-3’ Internal BAC sequence corresponding to the human COL8A2 gene: F: 5'-CTG AGA AGA AAG GTG CCA AC-3' R: 5'-CAG GAG CTG ATG AAT TTT CG-3' Chromosome location: Equine metaphase spreads for fluorescence in situ hybridisation (FISH) on GTG-banded chromosomes were prepared using pokeweed mitogen stimulated blood lymphocytes. BAC clone CHORI241-184G10 was digoxigenin labelled by nick translation using a nick translation mix (Roche Diagnostics, Mannheim, Germany). FISH on the GTG-banded horse chromosomes was performed using 750 ng digoxigenin-labelled BAC DNA, and 20 µg sheared total equine DNA and 10 µg salmon sperm DNA as competitors. After hybridisation overnight, signal detection was performed using a rhodamin detection kit (Qbiogene, Heidelberg, Germany). The chromosomes were counterstained with DAPI (4’,6’-

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diaminidino-2-phenylindole) and embedded in Vectashield Mounting Medium (Vector Laboratories, Burlingame, USA). Metaphase chromosomes that had been previously photographed with a CCD camera were re-examined after hybridisation with a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) equipped for fluorescence. Chromosomes were identified according to the international system for chromosome nomenclature of the domestic horse (ISCNH 1997)5. The equine genomic BAC clone containing the COL8A2 gene was most precisely located to ECA2p15-p16 on metaphase chromosomes of 30 cells (Fig.1). Radiation hybrid mapping: To confirm the chromosomal location of the BAC clones, the Texas A&M University equine 5,000-rad hybrid panel6 (http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) was used to map the sequence tagged site (STS) marker of the SP6 BAC end located approximately 181 kb upstream of COL8A2. PCR reactions were performed in a total of 20 µl using either 25 ng RH cell line DNA or 25 ng DNA from BAC clone CH241-184G10, 15 pmol of each primer and 0.75 U Taq polymerase (Qbiogene, Heidelberg, Germany). Samples were denatured at 94 °C for 4 min followed by 35 cycles under the following conditions: denaturation for 30 s at 94 °C, annealing for 60 s at 60 °C, and extension for 40 s at 72 °C. The PCR was completed with a final cooling at 4 °C for 10 min. PCR products were separated on a 1.5% agarose gel. The presence and absence of a PCR product were scored and the results used for a two-point analysis with RHMAPPER-1.227 to find markers co-segregating with COL8A2 on the equine 8 RH5,000 panel . The retention frequency of the sequence tagged site (STS) marker of the SP6 BAC end sequence was 14.1% and the two-point analysis revealed linkage of CHORI241-184G10_SP6 at a distance of 22.06 cR to the RBBP4 gene. The corresponding LOD score was 12. The RBBP4 gene had been previously located by RH mapping at 0 cR on ECARH02b8 as well as on HSA1p35.1 at 31,785,917 bp to 32,815,347 bp (human genome map viewer build 35.1). Comment: The RH and FISH mapping results of the equine COL8A2 gene on ECA2p15-p16 agree with comparative mapping of the current equine-human comparative RH and cytogenetic map8 of ECA2p, which shows conserved synteny to HSA1p.

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Acknowledgements: This study was supported by grants of the German Research Council, DFG, Bonn (DI 333/12-1).

References 1 Muragaki Y. et al. (1991) J Biol Chem 266, 7721-7. 2 Biswas S. et al. (2001) Hum Mol Genet 10, 2415-23. 3 Perala M. et al. (1993) FEBS Lett 319, 177-80. 4 Warman M. L. et al. (1994) Genomics 23,158-62. 5 Bowling A. T. et al. (1997) Chromosome Res 5, 433-43. 6 Chowdhary B. P. et al. (2002) Mamm Genome 13, 89-94. 7 Slonim D. et al. (1997) J Comp Biol 4, 487-504. 8 Chowdhary B. P. et al. (2003) Genome Res 103, 742-51.

Figure 1 Chromosomal assignment of the equine BAC clone CH241-184G10 containing the COL8A2 gene by FISH analysis. (A) GTG-banded horse metaphase spread. (B) Double signals visible on ECA2 are indicated by arrows.

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Chapter 8

Assignment of the TYK2 gene to equine chromosome 7q12- q13 by FISH and confirmation by RH mapping

Dorothee Stübs

Assignment of the TYK2 gene to equine chromosome 7q12-q13 by FISH and confirmation by RH mapping

Assignment of the TYK2 gene to equine chromosome 7q12- q13 by FISH and confirmation by RH mapping

Background: The tyrosine kinase 2 gene (TYK2) is a member of the janus kinase gene family and encodes an 1187 amino acid protein. All four members of the janus kinase family JAK1, JAK2, JAK3, and TYK2 associate with various cytokine receptors and mediate the signal transduction by tyrosine phosphorylation of downstream targets (YAMOAKA et al., 2004). In addition to its signaling function in humans, TYK2 is also necessary for the correct localization of the interferon receptor 1 (IFNAR1) in the cell membrane (RAGIMBEAU et al., 2003). Studies with Tyk2 deficient mice demonstrated that these mice are viable and show very specific changes in several cytokine responses. Surprisingly, interferon a/ß signaling is only impaired but not completely abolished in these mice (KARAGHIOSOFF et al., 2003). Mutations in the murine Tyk2 gene are associated with increased susceptibility to infectious and autoimmune diseases (SHAW et al., 2003). The human TYK2 gene consists of 25 exons spanning 30003 bp on human chromosome 19p13.2 starting at 10,322,209 bp and ending at 10,352,211 bp (International Human Genome Sequencing Consortium, 2004). The objective of this study was to determine the chromosomal location of TYK2 in the horse by FISH and RH mapping.

Procedure: BAC library screening/sequence analysis: The equine BAC library CHORI-241 was screened to isolate a genomic BAC clone containing the TYK2 gene. High density BAC colony filters were probed according to the CHORI protocols (http://bacpac.chori.org) with a heterologous 32P-labelled insert of a human TYK2 cDNA clone (IRALp962L0830) provided by the Resource Center/Primary Database of the German Human Genome Project (http://www.rzpd.de/). An equine genomic BAC clone designated CH241-352C1 with an insert of approximately 100 kb containing the TYK2 gene was identified. BAC DNA was prepared from the BAC clone using the Qiagen plasmid midi kit (Qiagen, Hilden, Germany). BAC DNA

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sequences were obtained using the ThermoSequenase kit (AmershamBiosciences, Freiburg, Germany) and a LI-COR 4200 automated sequencer (LI-COR, Inc., Lincoln, Nebr., USA). A BLASTN sequence comparison of the equine SP6 BAC end sequence with the build 35.1 of the human genome sequence revealed a significant match (BLAST E-value 6.0e-43) over 196 bp (identity = 87%) starting at 10,606,427 bp of HSA19p13.2, approximately 246 kb downstream of human TYK2. Chromosomal location: The equine BAC clone was labelled with digoxygenin by nick translation using a nick-translation mix (Roche Diagnostics, Mannheim, Germany). FISH on GTG-banded horse chromosomes was performed using 500 ng of digoxygenin labelled BAC DNA. 20 µg sheared total equine DNA and 10 µg salmon sperm DNA were used as competitors in this experiment. After hybridization overnight, signal detection was performed using a Digoxygenin-FITC Detection Kit (Qbiogene, Heidelberg, Germany). The chromosomes were counterstained with DAPI (4’,6’-diaminidino-2-phenylindole) and embedded in propidium iodide/antifade. Metaphase chromosomes that had been previously photographed by using a highly sensitive CCD camera were re-examined after hybridization with a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) equipped for fluorescence. Identification of chromosomes followed strictly the international system for chromosome nomenclature of domestic horses (ISCNH 1997). Primer sequences: Primers for PCR amplification of a 249 bp fragment were designed from the CH241-352C1 SP6 BAC end sequence using the PRIMER3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). F 5’-ATC ACT GTA GGG GGC AGA GA-3’ R 5’-CTG GTG TCT CTG TGC AGG AA-3’ Radiation hybrid (RH) mapping/PCR conditions: To confirm the cytogenetic assignment the 5,000 rad TAMU equine radiation hybrid panel (CHOWDHARY et al., 2003) was used to map TYK2. PCR was carried out in a 20 µl reaction containing 25 ng of RH cell line DNA, 10 pmol of each primer and 0.85 U Taq polymerase (Qbiogene, Heidelberg, Germany). The reaction conditions started with a denaturing step at 94°C for 5 min followed by 34 cycles using the following protocol: denaturation for 45 s at 94°C, annealing for 45 s at 60°C and extension for 5 min at

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72°C. The PCR was completed with a final cooling at 4°C for 5 min. PCR products were separated on a 1.5% agarose gel. After scoring positive signals, a two-point analysis (http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) was conducted to find associations between TYK2 versus the markers of the first generation whole genome equine RH map.

Results: The equine genomic BAC clone CH241-352C1 containing the TYK2 gene was located to ECA 7q12-q13 by examination of metaphase chromosomes of 40 cells (Fig. 1). The sequence tagged site (STS) markers showed a retention frequency of 10.9% and the RH mapping revealed close linkage to HTG33 (6.19 cR; LOD >3.0). The linked microsatellite marker had been previously mapped on ECA 7 by radiation hybrid mapping (CHOWDHARY et al., 2003). The RH result confirms the result obtained by FISH. The physical assignment of the equine TYK2 gene on ECA7q12-q13 did not agree with the comparative mapping data of the current equine-human comparative map of the centromeric region of ECA7p, which showed conserved synteny to HSA11 (CHOWDHARY et al., 2003; MILENKOVIC et al., 2002). Synteny detected here, however, is in agreement with the actual comparative cytogenetic map (PERROCHEAU et al., 2006).

Acknowledgements: This study was supported by grants of the German Research Council, DFG, Bonn (DI 333/12-1).

References CHOWDHARY, B.P. et al.: The first-generation whole-genome radiation hybrid map in the horse identifies conserved segments in human and mouse genomes. Genome Res., 13, (2003), 742-751

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International Human Genome Sequencing Consortium: Finishing the euchromatic sequence of the human genome. Nature 431 (2004), 931-945 KARAGHIOSOFF, M. et al.: Central role for type 1 interferons and Tyk2 in lipopolysaccharide-induced endotoxin shock. Nat. Immunol. 4 (2003), 471-477 MILENKOVIC, D. et al.: Cytogenetic localization of 136 genes in the horse: Comparative mapping with the human genome. Mamm. Genome 13, (2002), 524-34

RAGIMBEAU J. et al.: The tyrosine kinase Tyk2 controls IFNAR1 cell surface expression. EMBO J. 22 (2003) 3, 537-547. SHAW, M. H. et al.: A natural mutation in the Tyk2 pseudokinase domain underlies altered susceptibility of B10.Q/J mice to infection and autoimmunity. Proc. Natl. Acad. Sci. USA 100 (2003), 11594-11599 YAMAOKA, K. et al.: The Janus kinases (Jaks). Genome Biology 5 (2004) 253

Figure 1 Chromosomal assignment of the equine BAC containing TYK2 by FISH analysis. G-banded metaphase spread before (left) and after (right) hybridization. Double signals indicated by arrows are visible on both equine chromosomes.

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Chapter 9

Assignment of CART1, COL9A3, GNRH2 and TCIRG1 to equine chromosomes by FISH and confirmation by RH mapping

Dorothee Stübs

Assignment of CART1, COL9A3, GNRH2 and TCIRG1 to equine chromosomes by FISH and confirmation by RH mapping

Assignment of CART1, COL9A3, GNRH2 and TCIRG1 to equine chromosomes by FISH and confirmation by RH mapping

Background: Source/description: The cartilage paired-class homeoprotein 1 gene (CART1) maps to HSA12q21.3-q22 starting at 84,176,353 bp and ending at 84,198,027 bp (GORDON et al., 1996). It encodes a 326-amino acid homeoprotein containing a paired-like domain that is expressed in the forebrain mesenchyme, branchial arches, limb buds and cartilages during embryogenesis in rodents. It may also be involved in development of the cervix (ZHAO et al., 1993). In humans the specific functions of the CART1 gene are not yet known. The human gene consists of 4 exons spanning 21675 bp (GORDON et al., 1996). Mutations in the CART1 gene are associated in mice with neural tube defects such as acrania and meroanencephaly. Cart1- homozygous mutant mice are born alive with acrania and meroanencephaly but die soon after birth. As the phenotype observed here resembles strikingly a corresponding human syndrome caused by a neural tube closure defect, these mice may provide a useful animal model for developing therapeutic protocols for neural tube defects (ZHAO et al., 1996). The collagen, type IX, alpha 3 (COL9A3) maps to HSA20q13.3 starting at 60,918,832 and ending at 60,942,955 (TILLER et al., 1998). It encodes alpha 3 type IX collagen with 684 amino acids, one of the three alpha chains of type IX collagen, the major collagen component of hyaline cartilage (BREWTON et al., 1995). The human gene consists of 28 exons spanning 24124 bp (DELOUKAS et al., 2001). Type IX collagen is a heterotrimeric molecule, which is usually found in tissues containing type II collagen, a fibrillar collagen (MATSUI et al., 2003). Splice site mutations in the COL9A3 gene are associated with multiple epiphyseal dysplasia (MED) (PAASSILTA et al., 1999; BONNEMANN et al., 2000; LOHINIVA et al., 2000). The autosomal dominantly inherited MED is characterized by knee pain, stiffness and difficulty walking during childhood. Some patients develop hip arthrosis and require surgical

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hip replacement after age 50 years. A splice acceptor mutation in intron 2 of the COL9A3 gene causes autosomal dominant MED affecting predominantly the knee joints and a mild proximal myopathy. An arg103-to-trp (trp3 allele) substitution in the COL9A3 gene is supposed to increase the risk of lumbar disc disease about 3-fold (PAASSILTA et al., 2001). In addition to the hypothalamic decapeptide gonadotropin-releasing hormone 1 (GNRH1) regulating reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes, many vertebrate species express a second GNRH form, GNRH2(WHITE et al., 1998). GNRH2 shares about 70% homology with the mammalian hypothalamic neurohormone GNRH1, the primary regulator of reproduction, but is encoded by a different gene. The gonadotropin-releasing hormone 2 gene (GNRH2) maps to HSA20p13 starting at 2,972,268 and ending at 2,974,391 (International Human Genome Sequencing Consortium, 2004) and encodes a 120 amino acid protein that is expressed in the midbrain (KASTEN et al., 1996) and, in contrast to GNRH1, up to 30-fold higher levels outside the brain, particularly in the kidney, bone marrow, and prostate (PAASSILTA et al., 2001). The human gene consists of 4 exons spanning 2124 bp (DELOUKAS et al., 2001). Exposure of normal or cancerous human or mouse T cells to GNRH2 triggered de novo gene transcription and cell-surface expression of a 67-kD non-integrin laminin receptor that is involved in cellular adhesion and migration and in tumor invasion and metastasis. Furthermore, GNRH2 also induced adhesion to laminin and chemotaxis toward SDF-1alpha, and augmented entry in vivo of metastatic T-lymphoma into the spleen and bone marrow, which may be of clinical relevance (CHEN et al., 2002). The T-cell immune response regulator 1 gene (TCIRG1) maps to HSA11q13.4-q13.5 starting at 67,563,059 bp and ending at 67,574,942 bp and consists of 20 exons spanning 11884 bp. Through alternate splicing, this gene encodes the proteins TIRC7 (essential in T-cell activation) and OC116 (osteoclast-specific subunit of the vacuolar proton pump) with similarity to subunits of the vacuolar ATPase (V- ATPase). V-ATPase is a multisubunit enzyme that mediates acidification of eukaryotic intracellular organelles. V-ATPase is comprised of a cytosolic V1 domain and a transmembrane V0 domain. TIRC7 contains 15 exons spanning 7.9 kb,

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whereas OC116 contains 20 exons with the last 14 introns and exons being identical to TIRC7. The encoded proteins seem to have different functions. In autosomal recessive osteopetrosis patients TCIRG1 is mutated (SOBACCHI et al., 2001; SCIMEKA et al., 2003). Inactivation of the Tcirg1 gene in mice caused osteoclast-rich osteopetrosis (LI et al., 1999).

Procedure: BAC library screening/sequence analysis: For isolating BAC clones containing the selected genes, the equine CHORI-241 BAC library was screened. High density BAC colony filters were probed according to the CHORI protocols (http://bacpac.chori.org) with heterologous 32P-labelled inserts of the respective human cDNA IMAGE clones provided by the Resource Center/Primary Database of the German Human Genome Project (http://www.rzpd.de/). BAC DNA was prepared from the positive BAC clones using the Qiagen plasmid midi kit (Qiagen, Hilden, Germany). BAC DNA end sequences were obtained using the ThermoSequenase kit (AmershamBiosciences, Freiburg, Germany) and a LI-COR 4200 automated sequencer (LI-COR Inc., Lincoln, Nebr., USA). Chromosomal location: The equine BAC clones were labelled with digoxygenin by nick translation using a nick-translation mix (Roche Diagnostics, Mannheim, Germany). For TCIRG1 and TYK2, FISH on GTG-banded horse chromosomes was performed using 500 ng digoxygenin labelled BAC DNA, and 20 µg sheared total equine DNA and 10 µg salmon sperm DNA as competitors. For the remaining genes, FISH was performed using 750 ng of digoxygenin labelled BAC DNA. 20 µg sheared total equine DNA and 10 µg salmon sperm DNA were used as competitors in these experiments. After hybridization overnight, signal detection was performed using a digoxygenin-FITC detection kit (Qbiogene, Heidelberg, Germany). The chromosomes were counterstained with DAPI (4’,6’-diaminidino-2-phenylindole) and propidium iodide and embedded in antifade. Metaphase chromosomes that had been previously photographed by using a highly sensitive CCD camera were re-examined after hybridization with a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) equipped

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for fluorescence. Identification of chromosomes followed strictly the international system for chromosome nomenclature of domestic horses (ISCNH 1997). Primer sequences: Primers for PCR amplification from the BAC sequences were designed using the

PRIMER3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The equine BAC sequences were then compared to the Build 35.1 of the human genome sequences with BLASTN. The results are given in Table 1. Radiation hybrid (RH) mapping/PCR conditions: To confirm the cytogenetic assignment the 5,000 rad TAMU equine radiation hybrid panel (CHOWDHARY et al., 2003) was used to map the respective genes. PCR was carried out in a 20 µl reaction containing 25 ng of RH cell line DNA, 10 pmol of each primer and 0.85 U Taq polymerase (Qbiogene, Heidelberg, Germany). The reaction conditions started with a denaturing step at 94°C for 5 min followed by 34 cycles using the following protocol: denaturation for 45 s at 94°C, annealing for 45 s at 60°C and extension for 5 min at 72°C. The PCR was completed with a final cooling at 4°C for 5 min. PCR products were separated on a 1.5% agarose gel. After scoring positive signals, a two-point analysis (http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) (SLONIM et al., 1997) was conducted to find associations between the selected genes and the markers of the first generation whole genome equine RH map (CHOWDHARY et al., 2003).

Results: The equine genomic BAC clone CH241-482I10 containing the CART1 gene was located to ECA28q13-q15 by examination of metaphase chromosomes of 30 cells (Fig. 1a). The COL9A3 including equine genomic BAC clone CH241-311A7 was located to ECA22q18-q19 by examination of metaphase chromosomes of 35 cells (Fig. 1b), examination of metaphase chromosomes of 30 cells located the equine genomic BAC clone CH241-33D15 containing the GNRH2 gene to ECA22q15 (Fig. 1c). The localisation of the equine genomic BAC clone CH241-526P8 containing the TCIRG1 gene was confirmed to ECA12q14 by examination of metaphase chromosomes of 35 cells (Fig. 1d).

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Radiation hybrid (RH) mapping: CART1: The retention frequency of the sequence tagged site (STS) markers was 16.3% and the RH mapping revealed close linkage to UM003 (2.12 cR; LOD >3.0). The linked microsatellite had been previously mapped at 9.1 cM from the beginning on ECA28 (GUERIN et al., 2003) and at 3.5 cR from HTG30 on ECARH28a (CHOWDHARY et al., 2003). COL9A3: STS-markers showed a retention frequency of 16.3% and the RH mapping revealed close linkage to SG19 (2.53 cR; LOD >3.0) for COL9A3. The linked microsatellite marker had been previously mapped on ECA22q19 by FISH (GODARD et al., 1997), by linkage mapping at 54 cM of ECA22 (GUERIN et al., 2003), and by radiation hybrid mapping at 285 cR from COR022 on ECARH22c (CHOWDHARY et al., 2003). GNRH2: The retention frequency shown by the STS-markers was 16.3% and the RH mapping revealed close linkage to UMNE077 (21.20 cR; LOD >3.0). The linked microsatellite marker had been previously mapped on ECA22 by radiation hybrid mapping (CHOWDHARY et al., 2003). TCIRG1: STS-markers showed a retention frequency of 28.3% and the RH mapping revealed close linkage to ADRBK1 (adrenergic receptor beta kinase 1) (20.83 cR; LOD >3.0) on ECA12. The STS-marker had been previously mapped on ECA12 at 164.8 cR by RH mapping (CHOWDHARY et al., 2003). The RH result confirmed the result obtained by FISH. ADRBK1 is located on HSA 11cen-q13 starting at 66,790,663 bp and ending at 66,810,945 bp (human genome map viewer build 35.1). In agreement with our human-equine comparative mapping results, the equine CHRM1 (muscarin acetylcholine receptor I) gene was also cytogenetically located on ECA12q14 (MILENKOVIC et al., 2002) and the human CHRM1 gene maps to HSA11q13 starting at 62,432,728 bp and ending at 62,445,588 bp (human genome map viewer build 35.1). The physical assignment of the equine genes to the horse chromosomes agreed with the comparative mapping data of the current equine-human comparative map (PERROCHEAU et al., 2006).

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Acknowledgements: This study was supported by grants of the German Research Council, DFG, Bonn (DI 333/12-1).

References: BREWTON, R.G. et al.: Molecular cloning of the alpha 3 chain of human type IX collagen: Linkage of the gene COL9A3 to chromosome 20q13.3. Genomics 30 (1995) 2, 329-336 BONNEMANN, C.G. et al.: A mutation in the alpha 3 chain of type IX collagen causes autosomal dominant multiple epiphyseal dysplasiawith mild myopathy. Proc. Nat. Acad. Sci. 97 (2000), 1212-1217 CHEN, A. et al.: The neuropeptides GnRH-II and GnRH-1 are produced by human T-cells and trigger laminin receptor gene expression, adhesion, chemotaxis and homing to specific organs. Nat. Med. 8 (2002), 1421-1426 CHOWDHARY, B.P. et al.: The first-generation whole-genome radiation hybrid map in the horse identifies conserved segments in human and mouse genomes. Genome Res., 13, (2003), 742-751 DELOUKAS, P. et al.: The DNA sequence and comparative analysis of human chromosome 20. Nature 414 (2001) 6866, 865-871 GODARD, S.: Characterization, genetic and physical mapping analysis of 36 horse plasmid and cosmid-derived microsatellites. Mamm. Genome, 8, (1997), 745-750 GORDON, D.F. et al.: Human Cart-1: structural organization, chromosomal localization, and functional analysis of a cartilage-specific homeodomain cDNA. DNA Cell Biol. 15 (1996) 7, 531-541

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GUERIN, G. et al.: The second generation of the International Equine Gene Mapping Workshop half- sibling linkage map. Anim. Genet., 34, (2003), 161-168 International Human Genome Sequencing Consortium: Finishing the euchromatic sequence of the human genome. Nature 431 (2004), 931-945 KASTEN, T.L. et al.: Characterization of two new preproGnRH mRNAs in the tree shrew: first direct evidence for mesencephalic GnRH gene expression in a placental mammal. Gen. Comp. Endocr. 104 (1996), 7-19 KARAGHIOSOFF, M. et al.: Central role for type 1 interferons and Tyk2 in lipopolysaccharide-induced endotoxin shock. Nat. Immunol. 4 (2003), 471-477 LI, Y.-P. et al.: Atp6i-deficient mice exhibit severe osteopetrosis due to lost of osteoclast- mediated extracellular acidification. Nat. Genet. 23 (1999), 447-451 LOHINIVA, J. et al.: Splicing mutations in the COL3 domain of collagen IX cause multiple epiphyseal dysplasia. Am. J. Med. Genet. 90 (2000), 216-222. MATSUI, Y. et al.: Matrix-deposition of tryptophan-containing allelic variants of type 9 collagen in developing human cartilage. Matrix Biol. 22 (2003), 123-129 MILENKOVIC, D. et al.: Cytogenetic localization of 136 genes in the horse: Comparative mapping with the human genome. Mamm. Genome 13, (2002), 524-34 PAASSILTA, P. et al.: COL9A3: A third locus for multiple epiphyseal dysplasia. Am. J. Hum. Genet. 64 (1999), 1036-1044 PAASSILTA, P. et al.: Identification of a novel common genetic risk factor for lumbar disk disease. J. Am. Med. Ass. 285 (2001), 1843-1849

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PERROCHEAU, M. et al.: Construction of a medium-density horse gene map. Anim. Genet., 37, (2006), 145-155 SCIMECA, J.C. et al.: Novel mutations in the TCIRG1 gene encoding the a 3 subunit of the vacuolar proton pump in patients affected by infantile malignant osteopetrosis. Hum. Mutat. 21 (2003), 151-157 SOBACCHI, C. et al.: The mutational spectrum of human malignant autosomal recessive osteopetrosis. Human Molecular Genetics 10 (2001) 17, 1767-1773 TILLER, G.E. et al.: Physical and linkage mapping of the gene for the alpha 3 chain of type IX collagen, COL9A3, to human chromosome 20q13.3. Cytogenet. Cell Genet. 81 (1998) 205-207 WHITE, R.B. et al.: Second gene for gonadotropin-releasing hormone in humans. Proc. Natl. Acad. Sci. USA 95 (1998) 1, 305-309 ZHAO, G.Q. et al. Cartilage homeoprotein 1, a homeoprotein selectively expressed in chondrocytes. Proc. Nat. Acad. Sci. USA 90 (1993) 8633-8637 ZHAO, Q. et al.: Prenatal folic acid treatment suppresses acrania and meroanencephaly in mice mutant for the Cart1 homeobox gene. Nat. Genet. 13 (1996) 275-283

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Figure 1 Chromosomal assignment of the equine BACs containing a) CART1, b) COL9A3, c) GNRH2, d) TCIRG1 by FISH analysis. G-banded metaphase spread before (left) and after (right) hybridization. Double signals indicated by arrows are visible on both equine chromosomes.

(a)

(b)

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(c)

(d)

- 116 - Assignment of CART1, COL9A3, GNRH2 and TCIRG1 to equine chromosomes by FISH and confirmation by RH mapping

Table 1 Selected genes, their human location and cDNA clone identity, insert size of the positive equine BAC clones, used BAC end sequence, Accession-no. of BAC end sequence, BLASTN sequence comparisons of equine BAC sequences with human genome.

Gene HSAa Human Human or murine BAC BAC end or location cDNA clone identity internal (bp) (IN) sequence (Build 35.1) insert size (kb) CHORI- 241- 12q21.3- 84,176,353 482I10 CART1 q22 84,198,027 IMAGp998K149747b 190 SP6 CHORI- 241- 60,918,832, 311A7 COL9A3 20q13.3 60,942,955 IRALp962H0327b 190 T7 CHORI- 241- 2,972,268, 33D15 GNRH2 20p13 2,974,391 IMAGp998P018507 180 SP6 CHORI- 241- 11q13.4- 67,563,059, 526P8 TCIRG1 q13.5 67,574,942 IMAGp998B1510243b 180 T7 a: HSA: homo sapiens autosome b: human cDNA clone c: murine cDNA clone

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Table 1 continued Gene BLAST Length of Identity Beginning Distance E-Value match (bp) of to (%) sequence selected alignment gene on HSA (kb) (bp)

CART1 1.0e-30 292 82% 84,045,774 131 COL9A3 2.4e-52 198 88% 60,828,742 94 GNRH2 1.0e-74 155 88% 2,903,818 68 TCIRG1 2.0e-1 741 93% 67,694,235 140 a: HSA: homo sapiens autosome b: human cDNA clone c: murine cDNA clone

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Chapter 10

General discussion

General discussion

General discussion There has been a great progress in the development of comparative gene maps between horse and man during the last years. However, while many studies concentrated on the development of genetic linkage maps or comparative physical maps using radiation hybrid mapping (Chowdhary et al. 2003), the determination of cytogenetical anchorage of mapped genes was rare. Meanwhile, the comparative equine-human gene map contains 713 genes, 441 on the RH map, 511 on the cytogenetic and 238 on both maps (Perrocheau et al. 2006). The most extensive comparative map developed from cytogenetically mapped genes existing at the beginning of this work was published by Milenkovic et al. (2002) and included 136 genes. The interval between the publication of this map and the next extensive publication of a cytogenetic comparative map was almost three years. The next extensive contribution to the cytogenetic map included the localization of 150 genes (Perrocheau et al. 2005). The actual comparative map of the horse is developed by Perrrocheau et al. (2006) and added 130 newly cytogenetically mapped BACs, so already 511 genes have been mapped cytogenetically today. Between these milestones of cytogenetical mapping several genes have been localized separately, especially immunity-related genes or genes concerning the bone and cartilage metabolism and expanded the cytogenetic map of the horse genome step by step as it is described in this work. The contribution to the comparative horse genome map data resulting from this thesis is the presentation of the mapping of 16 genes with previously unknown localization. The genes have been mapped cytogenetically by FISH and the results have been confirmed by radiation hybrid mapping afterwards. Linkage mapping had been performed for ATP2A2_MS5. At the beginning of this study, 17 genes have been chosen as candidate genes. Ten genes have been chosen as candidate genes for osteochondrosis: BGLAP, BMP2, CART1, COMP, GNRH2, POU1, PTHR1 and TYK2 have been selected due to their locations in the vicinity of an identified QTL derived from QTL studies for OC in the pig (Andersson-Eklund et al. 2000) as well as for their indicated role in the development of cartilage growth. COL9A3 has been chosen because mutations in

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this gene cause autosomal dominantly inherited multiple epiphyseal dysplasia (MED) in humans characterized by knee pain, stiffness and difficulty walking during childhood - similar symptoms as in horses with OC (Paassilata et al. 1999). In autosomal recessive osteopetrosis patients the picked candidate gene TCIRG1 is mutated (Scimeca et al. 2003). Five genes from different solute carrier families responsible for the production of several transporter or membrane molecules have been localized in this work. Mutations in these genes are accompanied by different disease patterns in humans. The human CHST4 gene encoding a protein that is involved in enzymatic synthesis of the disulfated disaccharide unit of corneal keratan sulfate (Akama et al. 2002) should also be mapped to the horse genome in this study. The last gene, ATP2A2, has been chosen as a candidate gene for chronic pastern dermatitis in coldblood horses. Mutations in this gene cause the autosomal dominantly inherited condition keratosis follicularis in men. All chosen candidate genes are of relevance for horse breeding, because they are related to bone or cartilage metabolism or may contribute to inherited diseases. Apart from two genes all localizations discovered for the candidate genes in this thesis were in agreement with the former existing equine-human comparative maps

(Milenkovic et al. 2002, Chowdhary et al. 2003) and reflected the expectations arising from those maps. One of the genes, SLC26A2, has only been localized by radiation hybrid panel mapping because the localization by FISH and RH was already published during this study by Brenig et al. (2004). Since there could not be found positive signals on all filters of the equine CHORI-241 BAC library for the POU1F1 gene, no BAC clone was ordered and the gene could not be mapped in this study. The COMP gene was expected to be localized on ECA10 or ECA7 according to the existing comparative gene maps. The gene was definitely mapped to ECA21q13 by FISH in this work. There had only been known synteny between ECA21q and HSA5 before, so the obtained result was in conflict with the existing comparative gene map. The localization to ECA21q13 was confirmed by radiation hybrid mapping, furthermore, so there has been shown previously unknown synteny between ECA21q and HSA19p for the first time.

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For the TYK2 gene, FISH and RH-mapping results were also in conflict with the existing comparative gene maps: TYK2 was mapped to ECA7q12-q13. There had only been known synteny between ECA7q and HSA19q before, so synteny between ECA7q and HSA19p has been detected in this work for the first time. In the actual comparative cytogenetic map (Perrocheau et al. 2006), TYK2 was not contained, but for another gene located in the centromeric region of ECA7q (NR1H2), synteny between ECA7q11 and HSA19q had been shown. So the result detected in this thesis was confirmed that there not only exists synteny between ECA7q and HSA19q, but also between ECA7q and HSA19p. Due to the possible influence of the mapped candidate genes for osteochondrosis, former QTL studies for osteochondrosis identified important regions for ECA2 and ECA4 and concentrated on those regions concerning the choice of candidate genes. The mapped genes create a useful pool of genes as a basis for future studies concerning the inheritance of diseases concerning the bone or cartilage metabolism in horses. As measured by the fact that the entire horse genome includes assumedly 20000 to 25000 genes, the contribution of 17 genes to the horse gene map seems not to be great at first sight. But the systematic RH-mapping of genomic sequences as it is done by some institutes at the moment was not the primary aim of this thesis. Furthermore, it has to be considered that there can be faults if genes or genomic sequences have only been RH-mapped. This thesis represents a further step towards the construction of physical and comparative maps of the horse genome, especially improving the physical anchorage by FISH mapping, before whole genome sequencing is likely to be completed. .

References Akama T.O., Misra A.K., Hindsgaul O., Fukuda N.M. (2002). Enzymatic synthesis in vitro of the disulfated disaccharide unit of corneal keratin sulfate. J. Biol. Chem 277: 42505-42513.

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Andersson-Eklund L., Uhlhorn H., Lundeheim N., Dalin G., Andersson L. (2000). Mapping quantitative trait loci for principal components of bone measurements and osteochondrosis scores in a wild boar x large wide intercross. Genet. Res. 75: 223-230. Brenig B., Beck J., Hall A.J., Broad T.E., Chowdhary B.P., Piumi F. (2004). Assignment of the equine solute carrier 26A2 gene (SLC26A2) to equine chromosome 14q15? q21 (ECA14q15? q21) by in situ hybridization and radiation hybrid panel mapping. Cytogenet. Genome Res. 107: 139. Chowdhary B.P., Raudsepp T., Kata S.R., Goh G., Millon L.V., Allan V., Piumi F., Guerin G., Swinburne J., Binns M, Lear T.L., Mickelson J., Murray J., Antczak

D.F., Womack J.E., Skow L.C. (2003). The first-generation whole-genome radiation hybrid map in the horse identifies conserved segments in human and mouse genomes. Genome Res. 13: 742-751. Milenkovic D., Oustry-Vaiman A., Lear T.L., Billault A., Mariat D., Piumi F., Schibler L., Cribiu E., Guerin G. (2002). Cytogenetic localization of 136 genes in the horse: Comparative mapping with the human genome. Mamm. Genome 13: 524- 34. Paasilata P., Lohiniva J., Annunen S., Bonaventure J., Le Merrer M., Pai L., Ala Kokko L. (1999). COL9A3: A third locus for multiple epiphyseal displasia. Am. J. Hum. Genet. 64: 1036-1044. Perrocheau M., Boutreux V., Chadi-Taourit S., Chowdhary B.P., Cribiu E.P., De Jong P.J., Durkin K., Hasegawa T., Hirota K., Ianuzzi L., Incarnato D., Lear T.L.,

Raudsepp T., Perucatti A., Di Meo G.P., Zhu B., Guerin G. (2005). Cytogenetic mapping of 150 genes on the horse genome. Plant and Animal Genome XIII Conference, Januar 15-19, 2005, San Diego, CA. Perrocheau M., Boutreux V., Chadi S., Mata X., Decaunes P., Raudsepp T., Durkin K., Incarnato D., Iannuzzi L., Lear T.L., Hirota K., Hasegawa T., Zhu B., De Jong P., Cribiu E.P., Chowdhary B.P., Guerin G. (2006). Construction of a medium- density horse gene map. Anim. Genet. 37: 145-155.

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Scimeca J.C., Quincey D., Parrinello H., Romatet D., Grosgeorge J., Gaudray P., Philip N., Fischer A., Carle G.F. (2003). Novel mutations in the TCIRG1 gene encoding the a3 subunit of the vacuolar proton pump in patients affected by infantile malignant osteopetrosis. Hum. Mutat. 21: 151-157.

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Chapter 11

Summary

Physical and comparative mapping of candidate genes on the horse genome

Dorothee Stübs

Summary

Physical and comparative mapping of candidate genes on the horse genome

The aim of this work was to create a contribution to the comparative physical gene map, especially to the cytogenetic gene map of the horse by mapping candidate genes using fluorescence in-situ hybridisation and radiation hybrid panel mapping. First of all, 17 genes have been chosen as candidate genes. All chosen candidate genes are of important relevance for the horse breeding, since they are all genes possibly playing an important role concerning the inheritance of different diseases. The genes BGLAP, BMP2, CART1, COMP, GNRH2, POU1F1, PTHR1, TCIRG1, TYK2 and COL9A3 have been chosen as candidate genes for osteochondrosis. Five genes from different solute carrier families, SLC1A3, SLC4A1, SLC9A5, SLC20A1 and SLC26A2, responsible for the production of several transporter or membrane molecules whose alterations are causing different inherited diseases in humans, have been localized in this work, too. Accessorily, the CHST4 gene encoding a protein being involved in in vitro enzymatic synthesis of the disulfated disaccharide unit of corneal sulphate and the ATP2A2 gene as candidate gene for pastern dermatitis, should also be mapped to the horse genome in this study. 15 genes were mapped cytogenetically by using FISH on GTG-banded horse chromosomes for the first time. One candidate gene, SLC26A2, has not been mapped by FISH, because the localization of this gene was published in 2004 and forestalled this study, when the gene was already RH-mapped but fluorescence in- situ hybridisation had still not taken place. For the candidate gene POU1F1, no orthologue equine BAC clone could be ordered, because there were no positive signals on all eleven filters of the CHORI-241 BAC library, so the gene could not be mapped. To confirm the cytogenetic assignment, the genes were localized by using the 5,000 rad TAMU equine radiation hybrid panel. A two-point analysis (http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) was conducted to find associations between the candidate genes and the markers of the first generation whole genome equine RH map. The results from RH-mapping confirmed the FISH-

- 129 - Summary

results in all cases. The microsatellite ATP2A2_MS5 was also mapped to the equine linkage map and corresponded to the results received by FISH and RH-mapping for the gene ATP2A2. For 14 genes, the results of the newly mapped genes reflected the expectations arising from the known human-equine comparative map. In two cases (genes COMP and TYK2), the results were in conflict with the former developed comparative map. So far, conserved synteny had only been detected for ECA21q and HAS5 as well as for ECA7q and HAS19q. In this thesis, the gene COMP was mapped to ECA21q13 by FISH and by RH- mapping, so conserved synteny between ECA21q and HAS19q has been detected for the first time. For the candidate gene TYK2, the equine localization found by FISH and RH-mapping was ECA7q12-q13, so conserved synteny between ECA7q and HSA19p has been shown for the first time with this work, too.

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Chapter 12

Erweiterte Zusammenfassung

Physikalische und vergleichende Kartierung von Kandidatengenen auf dem Pferdegenom

Dorothee Stübs

Erweiterte Zusammenfassung

Physikalische und vergleichende Kartierung von

Kandidatengenen im Pferdegenom

Einleitung Vor dem Hintergrund der Erforschung erblicher und züchterisch relevanter Krankheiten beim Pferd hat die Entwicklung der physikalischen Genkartierung beim Pferd und die Entwicklung vergleichender Genkarten zwischen Mensch und Pferd in den letzten Jahren große Fortschritte gemacht. Während die zytogenetische Kartierung neuer Genorte mittels FISH zu Beginn der Genomforschung beim Pferd im Vordergrund stand, expandiert die physikalische Kartierung mittels Radiation- Hybrid-Kartierung dank der Entwicklung des 5000 rad-Panels in den letzten Jahren stark. Die aktuelle vergleichende Genkarte umfasst 713 Gene, von denen 441 durch Radiation-Hybrid (RH)-Kartierung und 511 Gene mittels Fluoreszenz-in-situ- Hybridisierung (FISH) auf dem Pferdegenom lokalisiert wurden. 238 Gene wurden sowohl auf der zytogenetischen als auch auf der RH-Karte lokalisiert. Ziel dieser Arbeit ist es, einen Beitrag zur vergleichenden Genkarte zwischen Mensch und Pferd zu leisten und gleichzeitig durch die Kartierung sowohl mittels Fluoreszenz-in-situ-Hybridisierung als auch durch Radiation-Hybrid-Kartierung die zytogenetische Verankerung in der vergleichenden Genkarte zu verbessern. 16 Kandidatengene sollen in dieser Arbeit zum ersten Mal auf den physikalischen Genkarten beim Pferd lokalisiert werden.

Erweiterung der physikalischen Karten des Pferdes mittels Fluoreszenz-in-situ- Hybridisierung (FISH) und Radiation-Hybrid (RH)-Kartierung

Auswahl der Kandidatengene Insgesamt 17 Kandidatengene wurden für die physikalische Kartierung beim Pferd ausgewählt. Einen Schwerpunkt bei der Kandidatengenauswahl bildeten zehn Kandidatengene, die im Hinblick auf einen möglichen Zusammenhang mit Osteochondrose ausgewählt wurden.

- 133 - Erweiterte Zusammenfassung

Bei der Osteochondrose handelt es sich um eine Störung der enchondralen Ossifikation in den Gelenken junger Pferde, die sich durch Beeinträchtigungen im Bewegungsablauf, Gelenkschwellung und Schmerzen äußern kann. Häufig betroffen sind Fessel-, Sprung- und Kniegelenk, wobei auch Hüft-, Schulter- und Wirbelgelenke betroffen sein können. Charakteristische Merkmale sind subchondrale Knorpelfrakturen, subchondrale Knochenzysten, Knorpelusuren, Chondromalazie und das Auftreten von Knorpelschuppen. Kommt es zum Vorhandensein freier Knorpel- oder Knochenfragmente in einem Gelenk, wird die Krankheit als Osteochondrosis dissecans bezeichnet. Die Prävalenz von Osteochondrose beträgt bei verschiedenen Warmblutpferderassen zwischen 10 und 79,5%. Für das Auftreten der Erkrankung sind neben einer durch Heritabilitätsschätzungen belegten genetischen Komponente auch die Ernährung, Skelettwachstumsraten, hormonelle Einflüsse sowie Traumata von Bedeutung, sodass insgesamt von einem multifaktoriellen Geschehen ausgegangen werden kann. Die acht Kandidatengene BGLAP, BMP2, CART1, COMP, GNRH2, POU1, PTHR1 und TYK2 wurden auf Grund von QTL-Studien beim Schwein als möglicherweise am Auftreten der Krankheit beteiligte Gene ausgewählt. Zusätzlich wurden das Gen COL9A3, dessen Mutation beim Menschen für das Auftreten der autosomal dominant vererbten Krankheit Multiple epiphsäre Dysplasie (MED) verantwortlich ist, und das Gen TCIRG1, durch dessen Mutation es beim Menschen zum Krankheitsbild der autosomal rezessiv vererbten Osteopetrose kommt, als Kandidatengene für OC ausgewählt. Fünf Kandidatengene gehören unterschiedlichen Solute Carrier-Familien an (SLC1A3, SLC4A1, SLC9A5, SLC20A1 und SLC26A2) und codieren für diverse Transport- und Membranproteine. Mutationen in diesen Genen gehen mit unterschiedlichen Krankheitsbildern beim Menschen einher. So sind Mutationen des SLC1A3-Gens, das für ein Transportprotein an glutamatergen Synapsen codiert, beispielsweise mitbeteiligt am Auftreten von Alzheimer beim Menschen. Durch Veränderungen des SLC4A1-Gens kann es beim Menschen zum Auftreten zweier unterschiedlicher Krankheitsbilder kommen; außerdem sind Mutationen an diesem

- 134 - Erweiterte Zusammenfassung

Gen für die Sichelzellanämie in Malariaendemiegebieten verantwortlich. Das Protein, für das das Gen SLC9A5 verantwortlich ist, gewährleistet den Na-/H-Austausch der Zellmembranen. Das SLC20A1-Gen codiert für einen Natrium-abhängigen Phosphat- Transporter, der bei der Infektion des Menschen mit dem Leukämievirus der Gibbonaffen als Rezeptor für das Retrovirus dient. Als weiteres Kandidatengen wurde das Gen CHST4 ausgewählt, das für ein Protein codiert, das am enzymatischen Aufbau einer Untereinheit des Keratansulfats der Cornea beteiligt ist. Das Gen ATP2A2 wurde als Kandidatengen für Mauke ausgewählt. Veränderungen des ATP2A2 Gens lösen beim Menschen follikuläre Keratose aus.

Material und Methoden Isolierung equiner genomischer BAC-Klone Vom RZPD (German Human Resource Center/Primary Database) wurden cDNA IMAGE-Klone, die die Kandidatengene enthielten, bezogen. Diese wurden für die Suche nach BAC-Klonen, die die orthologen equinen Sequenzen enthielten, mit 32P markiert. Anschließend wurde eine radioaktive Hybridisierung mit den Filtern der equinen genomischen CHORI-241-Genbank durchgeführt. Nach Auswertung wurden für positive Signale die entsprechenden equinen genomischen BAC-Klone bestellt. Für das Gen POU1F1 konnten auf keinem der Filter positive Signale identifiziert werden, so dass für dieses Kandidatengen keine BAC-Klone bestellt werden konnten und keine weiteren Studien erfolgten. Die BAC-DNA wurde dann isoliert und die Länge der genomischen DNA-Fragmente durch Pulsfeldgelelektrophorese ermittelt. Anschließend erfolgte die Ansequenzierung der 3`- und 5`-Enden der BAC-Klone auf einem automatischen Sequenziergerät (LI-COR 4200 oder 400). Zur Bestätigung der Richtigkeit der Sequenzen wurden diese über das Programm BLASTN mit humanen Datenbankeinträgen verglichen. Zu Beginn der Arbeit erfolgte für die equinen genomischen BAC-Klone der Gene BMP2, COL9A3 und TCIRG1 zusätzlich eine ECL-Hybridisierung zur Verifizierung.

- 135 - Erweiterte Zusammenfassung

Fluoreszenz-in-situ-Hybridisierung Equine Blutlymphozyten wurden in einem Standard-Kulturmedium mit pokeweed- Mitogen stimuliert und 72 Stunden im Brutschrank bei 38 Grad Celsius kultiviert. Die Präparation der Metaphasechromosomen erfolgte nach zytogenetischen Standardmethoden. Im Folgenden wurden eine GTG-Bänderung der Chromosomen durchgeführt und gut gestreute Metaphasen fotografiert. Die DNA der equinen BAC-Klone wurde über Nick-Translation mit Digoxygenin markiert. Ihre Hybridisierung erfolgte über Nacht an den GTG-gebänderten equinen Chromosomen. Als Kompetitor-DNA zum Binden repetitiver Sequenzen wurden gescherte genomische equine DNA und Lachssperma-DNA verwendet. Mittels Digoxigenin-FITC Detektionskit wurden die Signale der hybridisierten Proben erfasst. Anschließend wurden die Chromosomen mit DAPI und Propidiumjodid gegengefärbt und in Antifade eingebettet. Danach erfolgte die Überprüfung auf Signale der vorher fotografierten Metaphasen und die Zuordnung zu den Metaphasenchromosomen gemäß internationaler Nomenklatur für die Pferdechromosomen (international system for chromosome nomenclature of domestic horses, ISCNDH).

Radiation Hybrid (RH)-Kartierung Für die Randsequenzen der equinen genomischen BAC-DNA wurden, wenn es signifikante Übereinstimmungen mit den jeweiligen orthologen humanen Genen gab, mit Hilfe der Primer3-Software Primersequenzen ermittelt. Für den orthologen BAC- Klon des Gens SLC1A3 wurde für die Randsequenzen kein signifikanter Treffer zum SLC1A3 Gen gefunden. Aus diesem Grund wurden eine Subklonierung des BAC- Klons mit EcoR1 und die Ansequenzierung der Randsequenzen der isolierten Subklon-DNA durchgeführt. Aus einer Subklon-Randsequenz mit signifikanter Übereinstimmung mit dem SLC1A3 Gen konnten so Primer generiert werden. Mittels PCR erfolgte dann die Typisierung des Primer-DNA-Fragments an den Zelllinien des equinen 5,000 rad Texas A&M University RH Panels. Anschließend wurde unter Verwendung von RHMAPPER -1.22 (http://equine.cvm.tamu.edu/cgi- bin/ecarhmapper.cgi) die Auswertung durch Zwei-Punkt-Analyse mit den 861

- 136 - Erweiterte Zusammenfassung

Markern der equinen RH-Karte, die von Chowdhary et al. (2003) entwickelt worden war, durchgeführt.

Ergebnisse Insgesamt wurden von den 17 Kandidatengenen, deren Lokalisation auf dem Pferdegenom vorher unbekannt war, 15 Gene mittels Fluoreszenz-in-situ- Hybridisierung kartiert und ihre Lokalisation mittels Radiation Hybrid-Kartierung bestätigt. Außerdem wurde die Lokalisation des im Gen ATP2A2 enthaltenen Mikrosatelliten auf der Linkage-Karte des Pferdes ermittelt. Die Lokalisation des Gens SLC26A6 wurde nur durch RH-Kartierung ermittelt, da noch vor Abschluss der Kartierung mittels FISH die Kartierung des Gens von anderen Autoren veröffentlicht wurde. Das Gen POU1F1 konnte nicht kartiert werden, da bei der radioaktiven Hybridisierung auf allen elf Filtern der equinen genomischen CHORI-241 BAC- Genbank keine Signale vorhanden waren. Die Ergebnisse der Kartierungen sind in Tabelle 1 zusammengefasst. Für 14 kartierte Gene wurden die Erwartungen, die aus den bekannten vergleichenden Genkarten von Milenkovic et al. (2002) und Chowdhary et al. (2003) resultierten, bestätigt. Die Lokalisationen der Gene COMP und TYK2 standen im Widerspruch zu diesen vergleichenden Genkarten: Das COMP Gen wurde auf ECA21q13 kartiert, das Gen TYK2 auf ECA7q12-q13. Mit diesen Ergebnissen wurden erstmals Syntäniebeziehungen zwischen ECA21q und HSA19p sowie zwischen ECA7q und HSA19p aufgezeigt.

Diskussion Für insgesamt 14 kartierte Gene entsprach die Lokalisation auf dem Pferdegenom den Erwartungen aus den zu diesem Zeitpunkt aktuellen vergleichenden Genkarten. Das Gen COMP, für das laut vergleichenden Genkarten eine Lokalisierung auf ECA7 oder 10 erwartet worden wäre, wurde durch FISH auf ECA21q13 kartiert. Vorher waren Synteniebeziehungen nur zwischen ECA21q und HSA5 gezeigt worden. Das Ergebnis der RH-Kartierung bestätigte jedoch die Lokalisation. Auch für das Gen TYK2 entsprach das Ergebnis der FISH-Kartierung nicht den gültigen vergleichenden

- 137 - Erweiterte Zusammenfassung

Genkarten: das Gen wurde auf ECA7q12-q13 kartiert. Bislang waren Syntäniebeziehungen nur zwischen ECA7q und HSA19q bekannt gewesen. Auch dieses Ergebnis wurde durch ein gleich lautendes Resultat der RH-Kartierung untermauert. In dieser Arbeit werden demzufolge erstmals neue Syntäniebeziehungen zwischen ECA21q und HSA19q sowie zwischen ECA7q und HSA19p aufgezeigt. Das Gen COMP ist in der aktuell gültigen vergleichenden Genkarte enthalten. Das Gen TYK2 ist in dieser vergleichenden Genkarte noch nicht enthalten, jedoch bestätigt die Genkarte ebenfalls die festgestellte Syntäniebeziehung zwischen ECA7q und HSA19p. Insgesamt steht also der Beitrag neuer kartierter Gene für die physikalischen und vergleichenden Genkarten beim Pferd als Ergebnis dieser Arbeit im Vordergrund. Außerdem wurden in dieser Studie erstmals Syntäniebeziehungen zwischen ECA21q und HSA19q sowie zwischen ECA7q und HSA19p festgestellt.

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Chapter 13

Appendix

Appendix

Laboratory paraphernalia Equipment Thermocycler PTC-100TM ProgrammableThermal Controller (MJ Research, Watertown, USA) PTC-100TM Peltier Thermal Cycler (MJ Research, Watertown, USA) PTC-200TM Peltier Thermal Cycler (MJ Research, Watertown, USA)

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 1000 (Ammersham Biosciences, Freiburg)

Centrifuges Sigma centrifuge 4-15 (Sigma Laborzentrifugen GmbH , Osterode) Desk-centrifuge 5415D (Eppendorf, Hamburg) Biofuge stratos (Heraeus, Osterode) Centrifuge Centrikon H 401 (Kontron, Gosheim) Megafuge 1. OR (Heraeus, Osterode) Speed Vac R Plus (Savant Instruments, Farmingdale, NY, USA)

Agarose gel electrophoresis and pulsed field gel electrophoresis Electrophoresis chambers OWL Separation Systems, Portsmouth, NH, USA Biometra Göttingen BioRad, München Generators 2301 Macrodrive 1 (LKB, Bromma, Sweden) Power Pack 3,000 (BioRad, München) Gel documentation system BioDocAnalyze 312 nm, Göttingen

Fluorescence-in-situ-hybridization Zeiss Axioplan 2 microscope (Carl Zeiss, Jena) SenSys Digital CCD Camera system, Kodak KAF 1400 (Fa. Photometrics, München)

Pipettes MultipetteR plus (Eppendorf, Hamburg)

- 141 - Appendix

PipetusR-akku (HirschmannR Laborgeräte GmbH & Co. KG, Eberstadt) PipetmanR (P2, P10, P20, P100, P200, P1000) (Gilson Medical Electronics S.A., Villiers-le-bel, France) Pipettor, Multi 12 Channel (0.1-10 µl) (MicronicR systems, Lelystad, The Netherlands) ImpactR Pipettor 8-Channel (Matrix Technologies Corporation, Lowell, USA) HAMILTON 8-channel syringe (Hamilton Company, Reno, NV, USA) DistrimanR (Abimed, Langenfeld)

Others Milli-QR biocel water purificationsystem (Millipore GmbH, Eschborn) Incubator VT 5042 (Heraeus, Osterode) UV-Illuminator 312 nm (Bachhofer, Reutlingen) CentomatR R Desk-Shaker (B. Braun Melsungen AG, Melsungen) Biophotometer (Eppendorf AG, Hamburg)

Kits FISH Dig-Nick Translation Mix (Roche Diagnostics, Mannheim, Germany) Digoxigenin-FITC Detection Kit (Qbiogene, Heidelberg, Germany) Rhodamin Detection Kit (Qbiogene, Heidelberg, Germany)

Isolation of DNA HiSpeed Plasmid MIDI Kit (QIAGEN, Hilden) Plasmid MIDI Kit (QIAGEN, Hilden) Plasmid Mini Prep 96 Kit (Millipore GmbH, Eschborn) Qiagen Large Construct Kit (QIAGEN, Hilden)

Radioactive Hybridization RadPrime DNA Labelling System (Gibco-BRL, Eggenstein)

DNA purification

Montage PCR96 Cleanup Kit (Millipore GmbH, Eschborn)

Sequencing ThermoSequenase Sequencing Kit (Amersham Biosciences, Freiburg, Germany)

- 142 - Appendix

DYEnamic-ET-Terminator Cycle Sequencing Kit (Amersham Biosciences, Freiburg,

Germany)

Size standards 100 bp Ladder (New England Biolabs, Schwalbach Taunus) 1 kb Ladder (New England Biolabs, Schwalbach Taunus) IRDyeTM 700 or 800 (LI-COR, Inc., Lincoln, NE, USA)

Reagents and buffers APS solution (10%) 1g APS

10 ml H2O Bromophenol blue solution 0.5 g bromophenol blue 10 ml 0.5 M EDTA solution

H2O ad 50 ml dNTP solution 100 µl dATP [100mM] 100 µl dCTP [100mM] 100 µl dGTP [100mM] 100 µl dTTP [100mM]

1600 µl H2O the concentration of each dNTP in the ready-to-use solution is 5 mM Gel solution 12.75 ml Urea/TBE solution (Roth, Karlsruhe) 2.25 ml Rotiphorese® Gel 40 (38% acrylamide and 2% bisacrylamide) 95 µl APS solution (10%) 9.5 µl TEMED High stringency wash buffer II

1 mM Na2EDTA

40 mM NaHPO4, pH 7.2 7% SDS

- 143 - Appendix

Hybridization solution II (Church buffer) 1% bovine serum albumine (BSA) 1 mM EDTA

0.5 M NaHPO4, pH 7.2 7% SDS 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) Loading buffer for gel electrophoresis 2ml bromophenol blue solution 20 ml formamide Low stringency wash buffer II 0.5% bovine serum albumine (BSA) 1 mM EDTA

40 mM NaHPO4, pH 7.2 5% SDS TBE-buffer (1x) 100 ml TBE-buffer (10x)

900 ml H2O TBE-buffer (10x) 108 g Tris [121.14 M] 55g boric acid [61.83 M] 7.44 g EDTA [372.24 M]

H2O ad 1000 ml pH 8.0 Wash buffer for FISH

100 ml 1 mol Na2HPO4

100 ml 1 mol NaH2PO4 1 ml Tween 20

- 144 - Appendix

Chemicals a-[32P]-dCTP (50 µl, 3,000 Ci/mmol) (Hartmann Analytic GmbH, Braunschweig) Agarose (Invitrogen, Paisley, UK) Ampicillin (Serva, Heidelberg) Boric acid = 99.8 %, p.a. (Carl Roth GmbH & Co, Karlsruhe) Bromophenol blue (Merck KgaA, Darmstadt) dATP, dCTP, dGTP, dTTP > 98 % (Carl Roth GmbH & Co, Karlsruhe) Chloramphenicol (Serva, Heidelberg) Colcemid (Biochrom AG, Berlin) DAPI (Sigma-Aldrich Chemie GmbH, Taufkirchen) Dinatriumhydrogenphosphat (Biochrom AG, Berlin) DMSO = 99.5 %, p.a. (Carl Roth GmbH & Co, Karlsruhe) dNTP-Mix (Qbiogene GmbH, Heidelberg) EDTA = 99 %, p.a. (Carl Roth GmbH & Co, Karlsruhe) Ethidium bromide (Carl Roth GmbH & Co, Karlsruhe) Ethyl alcohol (AppliChem, Darmstadt) FKS (Biochrom AG, Berlin) Formamide = 99.5 %, p.a. (Carl Roth GmbH & Co, Karlsruhe) LB (Luria Bertani) agar (Scharlau Microbiology, Barcelona, Spain) LB (Luria Bertani) broth (Scharlau Microbiology, Barcelona, Spain) Natriumdihydrogenphosphat (Biochrom AG, Berlin) Paraffin (Merck KgaA, Darmstadt) Penicillin/Streptomycin (Biochrom AG, Berlin) Pokeweed mitogen (Biochrom AG, Berlin) Propidiumjodid (Sigma-Aldrich Chemie GmbH, Taufkirchen) Rotiphorese® Gel 40 (Carl Roth GmbH & Co, Karlsruhe) RPMI 1640 (Biochrom AG, Berlin) Trypsin (Biochrom AG, Berlin) Tris PUFFERAN® = 99.9 % (Carl Roth GmbH & Co, Karlsruhe) Twee20 (Carl Roth GmbH & Co, Karlsruhe) Urea = 99.5 % (Carl Roth GmbH & Co, Karlsruhe)

- 145 - Appendix

Water was taken from the water purification system Milli-Q® X-Gal (AppliChem, Darmstadt)

Clones All required clones have been ordered at the Resource Center/Primary Database (RZPD, http://www.rzpd.de).

Enzymes Taq-DNA-Polymerase 5 U/µl (Qbiogene GmbH, Heidelberg)

Incubation-Mix (10 x) T.Pol with MgCl2 [1.5 mM] (Qbiogene GmbH, Heidelberg) The polymerase was always used in the presence of incubation Mix t.Pol 10 x buffer. The enzymes SAC1, HIND III and ECO R1 (New England Biolabs, Schwalbach Taunus) were used with the adequate 10 x enzyme buffer.

BAC-libraries and RH-panel The gene-bank-screening was carried out on the CHORI-241 Equine BAC-Library (http://bacpac.chori.org/equine241.htm). The RH-panel used was the Texas A & M University equine Radiation Hybrid Panel (Chowdhary et al. 2002 and 2003).

Consumables 96 Well Multiply PCR plates, skirted (Sarstedt, Nümbrecht) Centrifugal Tubes (Tierärztebedarf Lehneke, Schortens) Combitips® plus (Eppendorf AG, Hamburg) Microscope slide (Tierärztebedarf Lehneke, Schortens) Pipette Tipps 0.1-10 µl, 5-200 µl (Carl Roth GmbH & Co, Karlsruhe) Reaction tubes 1.5 and 2.0 ml (nerbe plus GmbH, Winsen/Luhe) Reaction tubes 10 and 50 ml (Falcon) (Renner, Darmstadt) Serological pipettes 2 and 10 ml (Biochrom AG, Berlin) Thermo-fast 96 well plate, skirted (Abgene, Hamburg) Tissue culture flasks (Biochrom AG, Berlin)

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X-ray films Kodak Biomax MS, developer and fixer (Eastman Kodak Company, Rochester, NY, USA)

Software BLASTN, trace archive http://www.ncbi.nlm.nih.gov HORSEMAP database http://dga.jouy.inra.fr/cgibin/lgbc/loci_ IPLab 2.2.3 Scanalytics, Inc Order of primers MWG Biotech-AG, Ebersberg (https://ecom.mwgdna.com/register/index.tcl) biomers.net GmbH, Ulm ([email protected]) Primer design http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi Purchase of BACs http://bacpac.chori.org/ Repeat Masker http://www.repeatmasker.genome.washington.edu/ RHMAPPER-1.22 http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi Slonim et al. 1997 Sequencher 4.2 GeneCodes, Ann Arbor, MI, US

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Chapter 14

List of publications

Publications

Journal articles

Müller D., Kuiper H., Mömke S., Böneker C., Drögemüller C., Chowdhary B.P. and Distl O. (2005). Assignment of the COMP gene to equine chromosome 21q12- q14 by FISH and confirmation by RH mapping. Animal Genetics 36: 277-279.

Müller D., Kuiper H., Böneker C., Mömke S., Drögemüller C., Chowdhary B.P. and Distl O. (2005). Physical mapping of the PTHR1 gene to equine chromosome 16q21.2. Animal Genetics 36: 282-284.

Müller D., Kuiper H., Böneker C., Mömke S., Drögemüller C., Chowdhary B.P. and Distl O. (2005). Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5 and SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping. Animal Genetics 36: 457-460.

Müller D., Kuiper H., Böneker C., Drögemüller C., Swinburne J.E., Binns M., Chowdhary B.P. and Distl O. (2006). Physical mapping of the ATP2A2 gene to equine chromosome 8p14->p12 by FISH and confirmation by linkage and RH mapping. Cytogenetic and Genome Research 114: 94.

Stübs D., and Distl O. (2006). Mapping the horse genome and its impact on equine genomics for identification of genes for monogenic and complex traits. Accepted for publication in Archives for Animal Breeding.

Böneker C., Müller D., Kuiper H., Drögemüller C., Chowdhary B.P. and Distl O.(2005). Assignment of the COL8A2 gene to equine chromosome 2p15-p16 by FISH and confirmation by RH mapping. Animal Genetics 36: 444-445.

- 151 - Acknowledgements

Acknowledgements

First of all, I want to thank Prof. Dr. Dr. habil. Ottmar Distl, the supervisor of my doctoral thesis, for offering me an interesting work and for giving me the opportunity to do the practical parts of this thesis before I finished my study of veterinary sciences. I am very thankful for his support, his patience, his humor and his bavarian dialect that always reminded me of my first semesters in Munic.

I also thank Prof. Dr. Cord Drögemüller and Prof. Dr. Tosso Leeb for their support and advice concerning scientific questions.

I am most thankful to Dr. Heidi Kuiper for making me acquainted with the scientific, especially with the cytogenetic working methods, for her scientific advice and her invaluable support, the many answered questions, the constructive criticism in the course of this work and for working and talking together in good athmosphere.

I wish to thank Flavia Martins-Wess, Simone Rak and Kathrin Löhring for helping me at the beginning of this work, and I am also thankful to Flavia for being a friend and the amusing aqua gym lessons we had.

I am very grateful to Dr. Stefanie Mömke, Dr. Anne Wöhlke, Dr. Claudia Dierks and Dr. Andreas Spötter for their support and their open ear for my questions.

I wish to thank Heike Klippert-Hasberg, Stefan Neander and Diana Seinige for their support and advice during the work in the laboratory.

My special thanks go to C. Mrusek and D. Böhm for their support in administrative questions and for tolerating my dog in the office.

Furthermore, I want to thank Jörn Wrede for his help when my computer tried to annoy me.

- 152 - Acknowledgements

I enjoyed working with so many friendly and ready to help persons at the Institute for Animal Breeding and Genetics. Thanks to all of you - it was a great time! I want to thank the clinic for horses for the cooperation concerning the horse blood samples and I am grateful to all horses donating their blood to expand the horse gene map.

Thanks to my brother Florian for helping me converting this file and for answering many questions concerning problems with the computer.

My very special thanks go to my beloved husband Oli for supporting me and especially for helping me attending to Finn during the last weeks writing this work in Wolgast – I know I was intolerable sometimes… I am thankful to Finn for all the minutes he gave me to write while he was sleeping. Last but not least I want to thank Indie for the relaxing promenades and for always making me happy.

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