syntenic mapping of human disease genes using animal models of autoimmunity

© 2000 Blackwell Science Ltd.

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As for most common diseases, susceptibility to autoimmunityis thought to be determined by both genetic and environ-mental factors. These diseases tend not to be inherited insimple Mendelian fashion but exhibit complex patterns ofsegregation. Their investigation can often be hampered byfactors such as late age at disease onset, variable penetrance,variable phenotypic expression (different combinations ofgenes may predispose to different patterns of disease), unknowngene–gene and gene–environment interactions, genetic hetero-geneity (different genes may produce the same phenotype)and misclassification of clinical phenotypes. In recent yearsconsiderable advances have been made in the investigationof the genetic basis of complex diseases. Initially this centredon using microsatellite-based linkage analysis of extendedaffected pedigrees or large collections of affected siblingpairs to conduct whole genome screening (WGS) (exclusionmapping) to identify novel chromosomal areas/genes linkedwith susceptibility. Linkage-based approaches have also beenused to examine the

a priori

hypothesis that possible candidatesusceptibility genes contribute to disease. Alternative strategiesinclude testing candidate genes directly using case–controlassociation studies, and are now becoming more popular,especially as a high-density single nucleotide polymorphism(SNP) map becomes available.

1

Identification of candidate disease-susceptibility genes canarise from several routes. First, it is possible that a WGSapproach may suggest chromosomal regions where physicaland genetic mapping studies have already identified a geneor genes which, from their known function, could be involvedin a relevant disease process. For example, linkage of type Iinsulin-dependent diabetes mellitus (IDDM) to a region con-

taining the insulin gene could suggest that this gene shouldbe targeted directly.

2

Secondly it is possible that what wealready know about disease pathology would suggest thedirect testing of a candidate gene. Such an example would betesting genes such as mannose binding lectin (

MBL

) and

Fc

γ

RII

in systemic lupus erythematosus (SLE) where they areboth known to be important factors for the removal of immunecomplexes.

3–4

Thirdly, it is possible to identify candidatesfrom gene expression studies. These may be able to suggestwhich genes are up or down regulated in disease states and,thus, which may be worth examining in genetic studies.Alternatively, it is possible that animal models of humanautoimmune disease can provide an important insight as towhich candidate genes should be studied. In this review wewill consider how data emerging from animal models of dis-ease can be used to identify candidate regions and genes forinvestigation of human autoimmune disease. In particular,we will address how comparative genome maps can suggestwhere human equivalents of an animal disease-susceptibilitylocus can be found using syntenic mapping.

Animal models of autoimmune disease

Animal models have provided a useful approach to under-standing the aetiology and pathology of several autoimmuneconditions. Some animal models of autoimmune disease arespontaneous, where the condition occurs in all or a propor-tion of animals. One such model is the nonobese diabetic(NOD) mouse where animals develop an autoimmune insulinitisand diabetes.

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Alternatively, models of autoimmunity havealso been developed where the disease can only be inducedin susceptible animals following an experimental proceduresuch as the administration of autoantigen or adjuvant. Thisis the case for experimental allergic encephalomyelitis (EAE),a mouse model for multiple sclerosis induced by the adminis-tration of myelin basic protein in adjuvant.

6

Animal modelsof autoimmune disease have been developed which reflecteither key or different aspects of autoimmune disease in humans.

Correspondence to

: Anne Barton, ARC-EU, Stopford Building, University of Manchester, Oxford Road, Manchester, M13 9PT, U.K. E-mail: [email protected]

Anne Barton is funded by the Medical Research Council, UK. All other authors are funded by the Arthritis and Rheumatism Campaign, UK.

Blackwell Science, LtdOxford, UKGSCGeneScreen1466-920XBlackwell Science, 2000112000008Syntenic mapping of humam disease genesA. Barton et al.

REVIEW

37Graphicraft Limited, Hong Kong

Syntenic mapping of human disease genes using animal models of autoimmunity

Anne Barton, Jane Worthington and W. E. R. Ollier

ARC-EU, Stopford Building, University of Manchester, Oxford Road, Manchester, M13 9PT, U.K.

Keywords

animal models, autoimmunity, comparative mapping, genetics.

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In most cases, the animal model is not an exact mirror of thedisease in humans although it is more likely to resemble thatin humans if the animal develops the disease spontaneously.Induced models tend to be strain specific. For example, DArats immunised with type II collagen develop an arthritis whereasF344 strain rats, immunised in the same way, do not.

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The animals most commonly used for disease models arerats and mice. This is because:• Considerable information is already known about their genetic makeup• They exhibit extensive genetic homology with humans• They are easy to breed and have a short gestation• Inbred and congenic strains can be defined that are disease susceptible or resistant• Cross and backcross experiments can be performed• Experiments can be easily replicated• Environmental exposures can be rigorously controlled

Animal strains have been highly inbred so that differentanimals from the same strain share the majority or largesections of their genomes. When selecting markers, such asmicrosatellite repeats, with which to investigate linkage,markers comprising alleles that are different between diseasepermissive and disease resistant strains can be chosen. There-fore, these highly inbred strains can function essentially asbi-allelic systems so that parental origin of alleles and identityby descent status in F

1

offspring can be unambiguously assigned.

WGS in animal models

In order to identify genetic regions controlling phenotypicdifferences between two inbred strains (one susceptible andone resistant) of an animal model, A and B, either a back-cross ( (A

×

B)F

1

×

B) or an intercross ( (A

×

B)F

1

×

(A

×

B)F

1

)is performed. Following the disease-triggering event, theprogeny are phenotyped for the disease or trait of interestand then genotyped using markers selected to discriminatebetween parental strain of origin and to be evenly spacedat 10–15 c

M

intervals along the genetic linkage map. Thephenotypes and genotypes are then correlated and linkageanalysis performed.

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Once an area of linkage is detected, thiscan be confirmed in a replication data set. The first genome-wide scan in an animal model of autoimmune disease wasperformed on a backcross of diabetes-susceptible NOD miceand diabetes-resistant B10-h-2

g7

mice using 53 microsatellitesand eight restriction fragment length variants dispersed through-out the mouse genome. Two diabetes-susceptibility loci out-side MHC were identified —

Idd-3

on mouse chromosome 3and

Idd-4

on mouse chromosome 11.

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Since this landmarkpublication a number of further WGS have been performedto identify diabetes-susceptibility loci in the NOD mouse.To date, at least 14 loci have been identified with themajor contribution being from the MHC.

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A similar strategyhas been applied to investigate the genetics of other animalmodels of autoimmune disease. For example, New Zealandblack (NZB) mice spontaneously develop glomerulonephritis

and haemolytic anaemia associated with the presence ofanti-DNA antibodies and serve therefore as a good animalmodel of systemic lupus erythematosus (SLE). New Zealandwhite (NZW) mice are phenotypically normal. A WGS of anF

2

intercross between NZB and NZW mice identified eightsusceptibility loci associated with antichromatin autoanti-body production, glomerulonephritis and/or mortality. OnlyMHC was linked to all three traits suggesting that differentphenotypic features of SLE may be determined by differentgenetic susceptibility factors.

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Syntenic mapping

Since the divergence of lineages from the primordialmammal, there has been reorganization of genome structureresulting from chromosomal rearrangements such as inver-sions and translocations. However, the number of rearrange-ments has been modest. For example, it has been estimatedthat only approximately 144 major rearrangements haveoccurred in the estimated 80 million years since the diver-gence of the lineages leading to humans and mice.

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As aresult, mammals share a large number of genes and many ofthese genes are arranged in the same order along areas of thechromosome. Hybridisation methods and physical mappingcan be used to compare the chromosomal location of homo-logous genes in different species and maps of conservedchromosomal segments can be obtained.

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Synteny is definedas the presence together on the same chromosome of two ormore gene loci whether or not in such proximity that theymay be subject to linkage. Conserved synteny refers to twoor more homologous genes that are syntenic in two ormore species regardless of the gene order whereas conservedlinkage refers to both synteny and gene order of homologousgenes between species.

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However, even in areas of conservedlinkage, small internal rearrangements can occur. For example,a 47-c

M

region of mouse chromosome 11 containing 62 genesis highly conserved with human chromosome 17 with theexception of just six genes.

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Comparative maps can be used to predict gene locationsin other species and can also be used to identify candidatedisease genes. They have been used successfully to identifygenes involved in single-gene disorders in humans based oninvolvement of the homologous gene in animal models.

14

However, perhaps more importantly, they can be used to aidthe investigation of complex disorders which are difficult tostudy in humans because of the reasons listed above.

Possible experimental strategies

A number of WGS for a variety of animal models of variousautoimmune diseases have now been published. Theseinclude a WGS of oil-induced arthritis (OIA),

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a rat modelof rheumatoid arthritis. Following injection of incompleteFreund’s adjuvant into the back, tail, foot pad or lymphnode of a susceptible rat, acute inflammation affecting the



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ankles, wrists, tarsal and IP joints occurs. The arthritis isusually self-limiting, peaking at 20–25 days then regressingslowly. Histologically, the inflammatory changes seen aresimilar to RA. Like RA, the disease is influenced by genderwith females having earlier onset of disease.

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OIA is known to have genetic predisposition and themajor locus determining susceptibility is

MHC

.

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In orderto exclude the effect of

MHC

and identify susceptibility locioutside this region, arthritis-prone DA rats were crossedwith

MHC

-identical arthritis-resistant LEW.1AV1 rats. AWGS of the F

2

intercross showed that markers in five areasdeviated from the expected allele frequencies. Two loci,nominated

Oia

2

and

Oia

3

, fulfilled the stringent criteriasuggested for claims of significant linkage of F

2

progeny.Both loci influence both incidence and severity of the disease.The susceptibility locus,

Oia 3

on rat chromosome 10,colocalises with a region linked to

CIA

in rats.

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The extent oflinkage in both models spans approximately 20 c

M

. By using

information about comparative mapping between humansand rats from databases available publicly (Table 1), it isapparent that this region of rat chromosome 10 is highlyconserved with the long arm of human chromosome 17. Inorder to identify the region in humans homologous to thearea of linkage in these rat models of inflammatory arthritis,the first step is to prepare a gene map of the linked region inthe animal model. Genes mapping to linked loci can be iden-tified from databases containing information on the rat ormouse genome, a number of which are available publicly(Table 2). With the recent publication of dense radiationhybrid maps for both the rat

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and mouse

19–20

genomes anda YAC-based physical map of the mouse genome,

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this taskhas been helped enormously. Over 500 genes have beenmapped in rats, most of the homologues of which have alsobeen mapped in humans and mice.

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In our example, genesthat map close to the peak of linkage in the CIA model of RAmap to 17q25 in humans. Databases containing information

Table 1 Examples of rat and mouse genetic databases available publicly through the World Wide Web

Database World Wide Web address

The Wellcome Trust, Oxford University http://www.well.ox.ac.uk/~bihoreau/key.htmlRat genetic database

The Wellcome Trust, Oxford University http://www.well.ox.ac.uk/rat_mapping_resources/rat_RH_framework_maps.htmlRat RH maps

The Whitehead Institute, MIT http://waldo.wi.mit.edu/rat/public/Genetic maps of the rat genome

Gothenburg University, Sweden http://ratmap.gen.gu.se/RATMAP

Laboratory for Genetic Research, Wisconsin, USA http://goliath.ifrc.mcw.edu/LGR/

Otsuka GEN Research Institute, http://www.otsuka.genome.ad.jp/ratmapOtsuka Pharmaceutical Co. Ltd, JapanOLETF project

Mouse Genome Database (MGD), http://www.informatics.jax.orgMouse Genome Informatics Web Site,The Jackson Laboratory, Bar Harbor, Maine

Rat Genome Data, http://www.informatics.jax.org/rat /Mouse Genome Informatics Web Site,The Jackson Laboratory, Bar Harbor, Maine

ARB Rat Genetic Database, http://www.nih.gov/niams/scientific /ratgbase/ratgbase.htmlNational Institute of Arthritis andMusculoskeletal and Skin Diseases

Table 2 Examples of human/rat/mouse homology genetic maps available publicly through the World Wide Web


The Wellcome Trust, Oxford University http://www.well.ox.ac.uk/rat_mapping_resources/#

Otsuka GEN Research Institute, http://ratmap.ims.u-tokyo.ac.jp/cgi-bin/comparative_home.plOtsuka Pharmaceutical Co. Ltd, JapanOLETF project

NCBI, Bethesda, USA http://www.ncbi.nlm.nih.gov/Homology/Human mouse homology database

The Wellcome Trust, Oxford University http://www.well.ox.ac.uk/rat_mapping_resources/rat_comparative99_maps.html


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about the human genome (Table 3) reveal that the regionharbours the strong candidate RA gene, tissue inhibitor ofmetalloproteinase-2 (

TIMP-2

). Genes that map close to theend of the area of linkage in the OIA model map to 17q 21–22, a region that also contains a number of candidate genesincluding protein kinase C

α

chain (

PKCA

). The distancebetween these two areas in the human genome equates to40 c

M

and the segment shows a high degree of homologywith the equivalent region of rat chromosome 10.

Having identified homologous region(s) in humans, avariety of approaches are available to test for linkage or asso-ciation with the disease under investigation. The choice ofapproach will depend on a number of factors including thetype of genetic material available, the presence of a strongcandidate gene and the size of the region to be investigated.For example, if a very strong candidate gene exists in thecandidate region, association studies can be performed usinga case-control approach. If the region to be investigated islarge, linkage studies may be more appropriate.

Advantages and disadvantages of using syntenic mapping approaches

The expectation is that animal models of disease will beexplained by the same disease processes as the human equi-valent and will thus provide for elucidation of the geneticbasis of disease in humans. However, there is some evidenceto suggest that this may not always be the case. For example,the MRL-Fas(lpr) mouse was long thought to be a goodmodel for SLE based on the development of complement-mediated glomerulonephritis and autoantibodies includingds-DNA antibodies. The molecular basis for the phenotypewas found to be a polymorphism in the

Fas

gene whichresulted in a defect in apoptosis.

22

However, studies of thegene in humans with SLE failed to demonstrate a similardefect. In fact, a polymorphism in the

Fas

gene leads toautoimmune lymphoproliferative syndrome, characterisedby hypergammaglobulinaemia, lymphadenopathy, spleno-megaly and systemic autoimmunity.

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Furthermore, there arenumerous examples where different genetic defects can resultin a similar clinical phenotype.

24–25

Is it worth thereforeextrapolating results of genetic studies in animal models to

humans? It is likely that studying animal models will help usto identify key pathways which are important in determin-ing susceptibility to a disease, but a single animal model isunlikely to be the panacea for genetic investigation of thecorresponding disease in humans. Even when a good animalmodel of autoimmune disease exists and a candidate gene isgenerated as a result of the model, the functional polymorph-ism may be different in humans. For example, linkage andassociation to the insulin gene in humans was demonstratedafter this gene was identified as a candidate diabetes genebased on the biology of IDDM and from studies in the NODmouse, where linkage and association were reported. However,in humans, the functional polymorphism lies in the VNTRregion

26

whereas the gene in NOD mice does not contain anequivalent polymorphism (Mouse genome database).

Comparative maps have been used successfully in identifyinglinkage in complex disease in humans syntenic to those regionslinked to animal models of disease. Examples of successfuluse of this strategy include the demonstration of linkage to

5p12-14

, syntenic to a murine EAE susceptibility locus, in21 Finnish families with multiple sclerosis.

27

Because a single animal model, even if the disease in animalsoccurs spontaneously, is unlikely to reflect all aspects of dis-ease in humans, the question is how best to use these modelsystems. One could look at one good model of disease inanimals and extrapolate results of genetic studies to humans,as has been done in the examples listed above. Alternatively,if a variety of models exist for the same disease, regions ofoverlap between models can be determined and used to generatecandidate disease genes/regions for investigation in humans,as in the case of the

Oia 3

/

Cia 5

locus of inflammatoryarthritis example. A third approach would be to identify anumber of animal models of different disease that might beexpected to share features or a genetic predisposition incommon with the human disease under investigation. Anexample of such an approach would be to generate autoimmunecandidate genes/regions from results of studies into a varietyof autoimmune diseases, including IDDM, multiple sclerosis,ulcerative colitis, Crohn’s disease and psoriasis. A recentcomparison of results of linkage studies in a variety of humanautoimmune diseases has shown that positive linkages mapnonrandomly into 18 distinct clusters.

28

This suggests that


Genethon, Evry, France http://www.genethon.fr/genethon_en.htmlHuman Genome Research Centre

NCBI, Bethesda, USA http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htmlOnline inheritance in man (OMIM)

NCBI, Bethesda, USA http://www.ncbi.nlm.nih.gov/genemap99/Genemap ’99

The Hospital for Sick Children, http://gdbwww.gdb.org/gdb/gdbtop.htmlToronto, Ontario CanadaGenome Database (GDB)

Table 3 Examples of human genetic databases available publicly through the World Wide Web



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genes exist which predispose to autoimmunity in general andthat other genes determine the susceptibility of the target organ.

It is now clear that both spontaneous and inducedanimal models of autoimmune disease can provide a powerfulapproach for identifying susceptibility genes. Recent advancesin bioinformatics and the construction of syntenic geneticmaps between species now allows a comparative approachto be taken, whereby the possible human homologous genescan be identified. Although it is unlikely that animal modelsof autoimmunity will have exactly the same genetic aetiologyas its human counterpart, this strategy is likely to determinegenetic defects in common pathological pathways. As such itprovides an important complementary approach to identify-ing human candidate disease genes.

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syntenic mapping of human disease genes using animal models of autoimmunity

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