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A Systematic Analysis of HumanDisease-Associated Gene SequencesIn Drosophila melanogasterLawrence T. Reiter,1 Lorraine Potocki,3 Sam Chien,2 Michael Gribskov,1,2

and Ethan Bier1,41Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0349, USA; 2SanDiego Supercomputer Center, University of California San Diego, La Jolla, California 92093, USA; 3Department of Molecularand Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA

We performed a systematic BLAST analysis of 929 human disease gene entries associated with at least onemutant allele in the Online Mendelian Inheritance in Man (OMIM) database against the recently completedgenome sequence of Drosophila melanogaster. The results of this search have been formatted as an updateable andsearchable on-line database called Homophila. Our analysis identified 714 distinct human disease genes (77% ofdisease genes searched) matching 548 unique Drosophila sequences, which we have summarized by diseasecategory. This breakdown into disease classes creates a picture of disease genes that are amenable to study usingDrosophila as the model organism. Of the 548 Drosophila genes related to human disease genes, 153 are associatedwith known mutant alleles and 56 more are tagged by P-element insertions in or near the gene. Examples ofhow to use the database to identify Drosophila genes related to human disease genes are presented. We anticipatethat cross-genomic analysis of human disease genes using the power of Drosophila second-site modifier screens willpromote interaction between human and Drosophila research groups, accelerating the understanding of thepathogenesis of human genetic disease. The Homophila database is available at http://homophila.sdsc.edu.

Studies in the fruit fly Drosophila melanogaster have al-tered our estimate of the evolutionary relationship be-tween vertebrate and invertebrate organisms. Key mo-lecular pathways required for the development of acomplex animal, such as patterning of the primarybody axes, organogenesis, wiring of a complex nervoussystem, and control of cell proliferation have beenhighly conserved since the evolutionary divergence offlies and humans. When these pathways are disruptedin either vertebrates or invertebrates, similar defects areoften observed. The utility of Drosophila as a modelorganism for the study of human genetic disease isnow well documented. Developmental defects such asthe mesenchymal malformations associated with Sae-thre-Chotzen syndrome (Howard et al. 1997), forma-tion of intracellular inclusions in polyglutamine-tractrepeat disorders such as spinocerebellar ataxia andHuntington disease (Fortini and Bonini 2000), and lossof cellular-growth control and malignancy resultingfrommutations of tumor suppressor genes (Potter et al.2000) have been analyzed effectively using Drosophilaas the model genetic system. The many basic processesthat are shared between Drosophila and humans, inconjunction with the recent completion of the Dro-

sophila genomic sequence, provide the necessary ingre-dients for launching systematic analyses of human dis-ease-causing genes in Drosophila. An important ques-tion that arises from the combination of this genomicinformation with the detailed mechanistic under-standing of many Drosophila genes is, which humandisease genes are most appropriate for study in Dro-sophila?

A survey of 289 Drosophila genes related to humandisease genes has been presented in the context of theDrosophila genome sequence release (Rubin et al. 2000)and subsequently by Fortini et al. (2000). Additionally,more focused studies of Drosophila ion-channel genes(Littleton and Ganetzky 2000) and cancer-gene relatedsequences (Potter et al. 2000) have been published.Here, we report on results generated by a cross-genomic analysis of the 929 Locuslink entries of hu-man disease genes known to have at least one mutantallele listed in the current version of the Online Men-delian Inheritance in Man (OMIM) (McKusick 2000)against the complete Drosophila genome sequence. Wecompiled this cross-genomic data into a databasecalled Homophila, which presently contains a set of714 clear-candidate human disease genes, their Dro-sophila counterparts (548 distinct genes), and any P-elements within 1 kb of these genes. This set of geneswas categorized by human disease type and existingmutant alleles of these genes were identified. Analysis

4Corresponding author.E-MAIL [email protected]; FAX (858) 822-2044.Article and publication are at www.genome.org/cgi/doi/10.1101/gr.169101.

Resource

1114 Genome Research 11:1114–1125 ©2001 by Cold Spring Harbor Laboratory Press ISSN 1088-9051/01 $5.00; www.genome.orgwww.genome.org

of this dataset and support material is currently avail-able via the World Wide Web in the form of a search-able cross-genomic database (Homophila, http://homophila.sdsc.edu), which has been designed to beautomatically updated as the number of disease-associated genes expands.

RESULTS

Development of Homophila as a Tool forCross-Genomic AnalysisAs a starting point for our analysis, we wished to de-termine which human disease genes have clearly re-lated counterparts in Drosophila. To this end, we ex-tracted the set of all known human-disease–associatedgenes from OMIM with Locuslink entries and com-pared this set of genes to the recently completed Dro-sophila genomic sequence (see Methods). By incorpo-rating information about both the human disease geneand its Drosophila counterpart, it is possible to querythe search results by key word, disease name, fly gene,and OMIM number. The outline of a typical query toHomophila is illustrated in Figure 1.

Identification and Analysis of Drosophila GenesRelated to Candidate Human Disease GenesUsing Homophila, we found that 714 of the 929 (77%)OMIM human disease gene entries have highly similar(E �10�10) cognates in Drosophila (Fig. 2), which werefer to as “related genes” hereafter. An E value of�10�10 indicates that the odds are <1 in 1010 that sucha match would happen by chance alone given the sizesof the two compared databases (e.g., OMIM Locuslinkentries and Flybase). We are aware that these Dro-sophila cognates may not be functional orthologs tothe human disease genes and are using the less-stringent term “related genes” to describe these similarsequences. It is notable, however, that even at a higherE-value cut-off, a significant fraction of human diseasegenes have matches in Drosophila (Fig. 2, e.g., >54%with E �10�40 and 29% with E �10�100). A list ofdisease phenotypes resulting from mutations in genesthat are highly related to Drosophila genes (E <10–10) isavailable as a separate table on the Homophila Website (Reiter et al. 2000) as the clear-hit list, and has beencategorized into various subclasses based on clinicalphenotype (Table 1). Because some of the 714 distincthuman disease genes match the same Drosophila-related sequences, the total number of different Dro-sophila counterparts of human clear-hit genes is 548distinct Drosophila genes. We found a large number ofhuman disease genes involved in nonmyelin-associated neurological disorders (74), cancer (79),skeletal disorders (26), and other developmental de-fects (35), as noted in previous studies. We also found

a large number of metabolic and storage disorders(160), which were not highly sampled categories ofgenes in prior surveys. Consistent with the prevalenceof disorders affecting metabolism and other generalcellular functions, 409 of the clear-hit human genes(e.g., 57%) also have cognates in yeast (e.g., E �10�10).An interesting feature of this inclusive data set, whichalso was not evident from the earlier analyses of moreselective sets of diseases, is the high-number of humangenes affecting the visual (43), cardiovascular (26), au-ditory (13), skeletal (26), and endocrine (50) systemswith Drosophila counterparts.

To determine what fraction of Drosophila clear-hitgenes already have been analyzed by loss-of-functiongenetics, we examined each entry in the list of 548cognates of human disease genes in the clear-hit listand searched for alleles of each of these genes system-atically using allele and gene tables available from Fly-base (Flybase 1999) (see Methods). In this manner, 153mutant alleles were identified (e.g., 28% of Drosophilaclear-hit genes). These alleles and the human disease-related sequences can be found on our Web site intabular form (Reiter et al. 2000).

A notable result of this allele analysis is that thegreat majority of Drosophila genes related to humandisease genes (e.g., 395 of 548) have not yet been ana-lyzed by loss-of-function genetics, which is consistentwith the finding that only 14% of the genes identifiedby the Drosophila genome project had been identifiedpreviously by individual researchers working on spe-cific hypothesis-driven projects (Rubin et al. 2000). Wethen determined what fraction of the 395 predictedDrosophila transcription units without known mutant

Figure 1 (continues on following page)

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alleles have P-elements inserted in or near them (e.g.,within 1 kb of the gene-coding region). By aligning themap positions for 3442 known P-element insertionslisted by the Berkeley genome project with the mappositions of the Drosophila genes related to humanclear-hit genes, we found 190 distinct P-element inser-tions that lie within or near disease-gene–related se-quences. When corrected for multiple insertions, these190 P-elements reduce to 102 distinct clear-hit Dro-sophila genes. Further analysis determined that 56 of

Figure 2 Number of Drosophila sequences related to humandisease genes as a function of E-value. A graph of the percent ofhuman disease genes with similar sequences in Drosophila as afunction of E-value. Black-filled bars indicate the percent of hu-man Locuslink entries (929 total) with matches to Drosophila se-quences. White-filled bars indicate the percent of unique Dro-sophila sequences that match one or more human disease genesequences. Note that even at E-values of �10�40, 54% of humandisease genes have matches to Drosophila sequences.

Table 1. (Continued)

Disorder No. of genes

Soft tissue 2Connective tissue 18Dermatologic 25Metabolic/mitochondrial 123Pharmacologic 12Peroxisomal 9Storage 37Glycogen storage 11Lipid storage 13Mucopolysaccaridosis 10Other 3

Pleitropic developmental 35Growth, immune, cancer 7Apoptosis 1Other 27

Complex other 9Total 714

Totals for categories of disease are in bold, subcategory totalsare in parenthesis, and individual categories are in plain text.

Table 1. Classification of 714 Clear-Hit DrosophilaGenes According to Human Disease Phenotypes

Disorder No. of genes

Neurological 74Neuromuscular 20Neuropsychiatric 9CNS/Developmental 8CNS/Ataxia 9Mental retardation 6Other 22

Endocrine 50Diabetes 10Other 40

Deafness 13Syndromic 7Nonsyndromic 6

Cardiovascular 26Cardiomyopathy 10Conduction defects 4Hypertension 7Atherosclerosis 3Vascular malformations 2

Ophthalmologic 43Anterior segment (13)Aniridia 1Rieger syndrome 1Mesenchymal dysgenisis 2Iridogoniodysgenisis 2Corneal dystrophy 2Cataract 3Glaucoma 2

Retina (30)Retinal dystrophy 1Choroiderimea 1Color vision defects 4Cone dystrophy 2Cone rod dystrophy 1Night blindness 8Leber congenital amaurosis 2Macular dystrophy 4Retinitis pigmentosa 7

Pulmonary 4Gastrointestinal 13Renal 13Immunological 33Complement mediated 11Other 22

Hematologic 42Erythrocyte, general 29Porphyrias 7Platelets 6

Coagulation abnormalities 28Malignancies 79Brain 3Breast 4Colon 11Other gastrointestinal 3Genitourinary 5Gynocologic 3Endocrine 3Dermatologic 3Xeroderma pigmentosa 6Other/sarcomas 9Hematologic malignancies 29

Skeletal development 26Craniosynostosis 5Skeletal dysplasia 13Other 8

Reiter et al.


these P-element insertions are the only known allelesof these genes. Using routine genetic methods in Dro-sophila, it should be possible to create null alleles of the56 P-element tagged genes with relatively little diffi-culty by remobilizing the P-elements and screening forimprecise excisions that delete all or parts of the cod-ing regions. Thus, loss-of-function analysis should bestraightforward for 209 (153 + 56) of the 548 clear-hitgenes, which represents a substantial proportion ofthese genes (e.g., 38%).

Defects at Multiple Tiers of ConservedSignal-Transduction Pathways Cause Human DiseaseTo explore the cross-genomic nature of the clear-hitgene dataset further, we subcategorized genes into oneof several signal transduction pathways known to playimportant developmental functions in Drosophila andlooked for trends in the resulting human phenotypes.Signal transduction pathways typically are activated byone or several ligands binding to one or more trans-membrane receptors. Ligand binding activates the re-ceptor and leads to modification of cytoplasmic trans-ducers that enters the nucleus-altering gene expres-sion. A feature common to many signaling pathways isthat multiple ligands activate specific receptors, whichconverge upon one or a few common cytoplasmictransducer(s).

Among the disease genes on the clear-hit list, 56(corresponding to 38 distinct Drosophila genes) encodecomponents acting in well-characterized signalingpathways such as the bone morphogenic protein(BMP), receptor tyrosine kinase/reticular activating sys-tem (RTK/RAS), G-coupled receptor, JAK/STAT, Toll,Integrin, and axonal-guidance pathways (Table 2). Sig-naling components in these pathways have been or-dered in Table 2 with respect to their position inknown signaling cascades in Drosophila (e.g., ligand->receptor->cytoplasmic transducer->transcription-factor effector). A notable trend apparent in these tabu-lated data is that mutations affecting particular ligandsor cell-type–specific receptors generally result in re-stricted developmental abnormalities in humans,whereas mutations in universally employed receptorsubunits or downstream intracellular signal transduc-ers tend to cause more global loss of cellular growthcontrol or cancer in humans. For example, in the caseof the BMP signaling pathway (Fig. 3), defects in spe-cific BMP ligands result in human bone malformation(e.g., brachydactyly) and mutations of a selective typeI BMP receptor subunit cause venous malformations(e.g., hereditary hemorrhagic telangiectasia). On theother hand, loss of the universal type II BMP receptorsubunit, or the core signal transducer (e.g., vertebrateSMAD4 = Drosophila Medea) results in cancer in hu-mans. This trend in which mutations in generally used

signaling components often lead to loss of cellulargrowth control and cancer is consistent with many sig-naling pathways being directly or indirectly involvedregulating cell proliferation.

DISCUSSIONOur goal in conducting the analysis described in thisstudy was to define a subset of human disease genesthat would benefit most frommolecular-genetic analy-sis in Drosophila. To this end we used Homophila, asearchable interactive database, to define a set of can-didate human disease genes that have clearly relatedgenes in Drosophila.

A strength of our current analysis with respect toprevious studies is that it is inclusive and encompassesa much larger nonredundant set of human diseasegenes listed in OMIM with Locuslink entries. In con-trast, previous studies have been more restrictive sur-veys focusing only on a subset of 289 genes selected apriori, which are known to be causally linked to a hu-man disease (Fortini et al. 2000; Rubin et al. 2000) orthose involved with a particular category of diseasestate (Littleton and Ganetzky 2000; Potter et al. 2000).This is a critical distinction because the current analy-sis reveals the relative proportions of different diseasesubclasses available for study in Drosophila. For ex-ample, in the previous two survey studies, genes affect-ing hearing and visual systems were relatively rare be-cause of the more restrictive selection criteria used. Ad-ditionally, we identified 123 metabolic genes (17% ofthose analyzed) whereas the previous studies only in-cluded 17 metabolic genes in the dataset (6% of thoseanalyzed).

Another problem with any type of cross-genomicanalysis is that one must determine which sequencematches are significant enough to be consideredsimilar in evolutionary origin. In addition, one mustbe able to distinguish domain-specific matches (e.g.,a cross-species match of leucine zipper domains)versus matches that span the entire amino-acid se-quence. For these reasons, we have provided a graphof the percent of human disease genes with Drosophilacounterparts at a variety of E-values (Fig. 2). We alsoimplemented a graphical interface for each match; thiswill provide the user with information about anno-tated domains of both the human and fly proteins (seeFig. 1).

Approximately Three-Quarters of the CandidateHuman Disease Genes are Clearly Related to Genesin DrosophilaAnalysis of the set of potential human disease genesrelated to Drosophila genes as defined in this study isinformative in several respects. First, we find a highprevalence of neurological and neurodegenerative con-



Table 2. Drosophila Genes From the Clear-Hit List That are in Known Signaling Pathways and the Human PhenotypesAssociated with These Disease Genes

Signaling pathway Disease OMIM# Fly gene Signaling component

BMP Fibrodysplasia ossificans progressiva 112262 (dpp) LigandBrachydactyly, type C 113100 (dpp) LigandAcromesomelic dysplasia,Hunter-Thompson type

601146 (dpp) Ligand

Hereditary hemorrhagictelangiectasia-2

601284 (sax) Specific type I receptor

Persistent Mullerian duct syndrome,type II

600956 (wit) Specific type II receptor

Colorectal cancer, familialnonpolyposis, type 6

190182 (put) General type II receptor

Polyposis, juvenile intestinal 174900 (med) Cytoplasmic transducerPancreatic cancer 600993 (med) Cytoplasmic transducer

Hedgehog Holoprosencephaly-3 600725 (hh) LigandBasal cell nevus syndrome 109400 (ptc) Co-receptorBasal cell carcinoma, sporadic 601309 (ptc) Co-receptorGreig cephalopolysyndactyly syndrome 165240 (ci) Transcription factor

Wnt Joubert syndrome 213300 (wg) LigandSimpson dysmorphia syndrome 300037 (dally) Proteoglycan (co-receptor?)Colorectal cancer 116806 (arm) Cytoplasmic transducer

Notch Alagille syndrome 601920 (Ser) LigandCerebral ateriopathy with subcorticalinfarcts and leukoencephalopathy

600276 (N) Receptor

RTK Obesity with impaired prohormoneprocessing

162150 (Fur1) Protease: Ligand activation?

Achondroplasia; Craniosynostosis;Crouzon syndrome

134934 (htl) Receptor

Pfeiffer syndrome 136350 (htl) ReceptorVenous malformations, multiplecutaneous and mucosal


Apert syndrome; Beare-Stevenson cutisgurata


Mast cell leukemia; Mastocytosis;Piebaldism


Diabetes mellitus, insulin-resistant;Leprechaunism; Rabson-Mendenhallsyndrome

147670 (InR) Receptor

Renal cell carcinoma 164860 Receptor kinase-like gene Receptor?Predisposition to myeloid malignancy 164770 Putative growth factor

receptorReceptor?

Bladder cancer 190020 (Ras85D) Cytoplasmic transducerColorectal adenoma 190070 (Ras85D) Cytoplasmic transducerColorectal cancer 164790 (Ras85D) Cytoplasmic transducerColon cancer 600679 Tyrosine phosphatase 99A PhosphataseEhlers-Danlos syndrome, type X 135600 Tyrosine phosphatase 10D PhosphataseElliptocytosis-1 130500 (cora) Cytoskeletal scaffolding?

Serpentine Hypertension, salt-resistant 108962 guanylate cyclase receptor ReceptorNight blindness, rhodopsin-related;Retinitis pigmentosa

180380 (ninaE) Receptor (Rhodopsin 1)

Colorblindness, deutan 303800 (ninaE) ReceptorRetinitis pigmentosa 4, included; rp4 180380 (ninaE) ReceptorNight blindness, congenital stationary,rhodopsin-related

190900 (ninaE) Receptor

Autonomic nervous system dysfunction 126452 Dopamine receptor-likegene

Receptor

Susceptibility to Schizophrenia? 126451 (DopR2) ReceptorNight blindness, congenital stationary,type 3

180072 cGMP phosphodiesterase Phosphodiesterase

Retinitis pigmentosa, autosomalrecessive

180071 cGMP phosphodiesterase Phosphodiesterase

Susceptibility to essential hypertension 139130 (Gbeta13F) Cytoplasmic transducerBleeding diathesis due to GNAQdeficiency

600998 (Galpha49B) Cytoplasmic transducer

JAK/STAT SCID, autosomal recessive,T-negative/B-positive type

600173 (hop) JAK kinase

Reiter et al.


ditions (Table 1). This finding is not entirely unantici-pated, because many of the components of neurogen-esis (such as factors involving neural induction, guid-ance cues leading axons to their appropriate targets,the machinery for generating and propagating actionpotentials, and enzymes and molecular complexes in-volved in the synthesis and release of neurotransmit-ters) have been highly conserved during the course ofevolution (Salzberg and Bellen 1996; Wu and Bellen1997). Within the category of neurological diseases,the relatively large number of hearing conditions isnoteworthy because these genes represent biologically

analogous systems (e.g., the hairs in the inner ear ver-sus the sensory bristles of Drosophila). Without thecomplete comparisons of the genomes in a databaselike Homophila, it would not be immediately obviousthat genes responsible for human deafness could befunctionally analyzed in an organism like Drosophila,which has no external auditory specializations analo-gous to ears. Second, we find that components of sig-nal transduction pathways are frequent targets of hu-man disease. An interesting relationship regarding thiscategory of disease genes is that mutations in differentcomponents of various signaling pathways can result

Figure 3 Relationship of a components position in the bone morphogenetic protein (BMP) pathway to human disease phenotypes. Ingeneral, there is a relationship between the position of a component in signaling pathway to the disease phenotype resulting frominactivation of that component. An example of this trend is the BMP pathway. Mutations in components acting at the start of the BMPsignal transduction cascade such as a particular BMP ligand (e.g., Drosophila Dpp = Human BMP4/BMP2) or a specialized BMP type Ireceptor (Drosophila Saxophone = type I receptor for the Screw and Glass Bottom Boat ligands) result in specific developmental defects(e.g., brachydactyly). Mutations acting on subsequent steps in the BMP pathway, which mediate the effects of several convergingupstream inputs such as the universal type II BMP receptor (e.g., Drosophila Punt = type II receptor mediating all BMP signaling) or thecytoplasmic/nuclear SMAD transducer (e.g., Drosophila Medea = Human SMAD4) result in generalized misregulation of cellular growthcontrol and cancer (e.g., colorectal or pancreatic cancer).

Table 2. (Continued)

Signaling pathway Disease OMIM# Fly gene Signaling component

Toll/NFkB Leukemia/lymphoma, B-cell 109560 (cact) Cytoplasmic transducerNF�I-like

Neuronal pathfinding Propedrin deficiency 312060 Semaphorin family Repulsive ligandPolycystic kidney disease, type I 601313 Slit-like gene Repulsive ligandAntithrombin III deficiency 107300 (sema-5c) Ligand?Transcortin deficiency 122500 (sema-5c) Ligand?Plasmin inhibitor deficiency 262850 (sema-5c) Ligand?Hydrocephalus due to aqueductalstenosis, MASA syndrome, spasticparaplegia

308840 (Nrg) Adhesion molecule(Neuroglian)

Colorectal cancer 120470 (fra) ReceptorIntegrin Glazmann thrombasthenia, type A 273800 (if) Integrin �-chain

Epidermolysis bullosa, junctional, withpyloric stenosis

147556 (mew) Integrin �-chain

Myopathy, congenital 600536 (mew) Integrin �-chainGlycoprotein Ia deficiency 192974 (mew) Integrin �-chain



in very different disease phenotypes in humans. Com-ponents acting at early stages in a given pathway, suchas genes encoding extracellular ligands, tend to havemore specific and limited phenotypes, while genes act-ing in more downstream capacities, such as those en-coding ligand receptors and downstream intracellularsignaling molecules exhibit broader sets of defects re-sulting from disruption of several converging upstreamsignals.

Loss-of-Function Genetics to Study Human DiseaseGenes In DrosophilaAnother striking feature of the list of potential humandisease genes with related genes in Drosophila (i.e., theclear-hit list) is that only a minority of these genesalready have been studied by classical loss-of-functiongenetics (i.e., 28% of the 548 Drosophila genes relatedto human disease genes on the clear-hit list). This num-ber highlights the substantial number of yet-unstudiedDrosophila cognates of human disease genes, whichcould be analyzed using the molecular genetic tools ofDrosophila. It should be possible to study the majorityof these genes using various previously-establishedmethods. For example, wild-type or disease-causingmutant variants of any candidate human disease genecan be misexpressed in Drosophila using routine meth-ods and the resulting gain-of-function phenotypes as-sayed either during development or in the adult. Be-cause developmental pathways have been extensivelystudied in Drosophila, observation of gain-of-functionphenotypes often will immediately implicate particu-lar candidate pathways. For example, in the Drosophilawing it is possible to distinguish phenotypes resultingfrom disruption of components in the EGF-Receptor(RTK), Notch, Wingless, Hedgehog, and Drosphila de-capentaplegic (Dpp) signaling pathways based on wingshape, integrity of the wing border, and the positionand number of wing veins. Isolation of a new mutantwith phenotypes resembling those of mutants in oneof these known pathways would suggest obvious fol-low-up experiments to verify that the new gene wasindeed involved in the suspected pathway. It also ispossible to assay neurobehavioral phenotypes in Dro-sophila such as defects in vision, chemosensation,touch, hearing, and rudimentary learning. The fewstudies of this kind that have been carried out to dateare very encouraging in that misexpression of diseasealleles of human genes often results in visible morpho-logical defects or behavioral deficits. A particularlypromising aspect of several of these studies is that thefunction of normal versus mutant alleles of the humandisease gene can be distinguished. A likely mechanisticbasis for the different activity of wild-type versus mu-tant forms of candidate human-disease genes is thatthe mutant may act as a dominant negative in Dro-

sophila as a result of a nonproductive interaction witha conserved component shared between flies and hu-mans.

The function of the endogenous Drosophila coun-terparts of candidate human disease genes also can beanalyzed by gain-of-function studies. More critically,however, loss-of-function analyses can be initiated todetermine the consequence of removing the activity ofthese genes in Drosophila. Such loss-of-function analy-sis can be carried out for any of the 56 yet-to-be-analyzed P-element tagged genes. Additionally, be-cause it is now practical to make targeted mutants inDrosophila (Rong and Golic 2000), it should soon befeasible to generate loss-of-function mutants in any ofthese genes. If misexpression of a human disease gene(normal or altered) or mutation of its Drosophila coun-terpart leads to scorable phenotypes in flies, second-site modifier screens typically can be designed to iden-tify further genetic components acting in the samepathway as the gene of interest. The use of Drosophilato identify second-site modifier loci, which can then betested for potential contribution to human disease (ormodification of disease phenotypes), is likely toemerge as the most valuable application of Drosophilaas model system for analysis of human disease genesbecause similar screens cannot be carried out on a sig-nificant scale in vertebrate systems.

Which Candidate Human Disease Genes are BestSuited for Analysis in Drosophila?The motivation for conducting the above analysis wasthe practical issue of identifying Drosophila genes re-lated to candidate human disease genes that are likelyto be productively studied in Drosophila. It is evidentthat not all genes on the clear-hit list are necessarilybest suited for study in Drosophila. For example, thegreat majority of the clear-hit human disease genes in-volved in metabolic and mitochondrial disorders (123)also have direct counterparts in yeast. Because many ofthese genes control similar basic cellular processes inyeast, flies, and humans, they may be more effectivelyanalyzed in yeast rather than in Drosophila. It is alsothe case that some genes common to Drosophila andhumans may not be performing equivalent functionsin these two organisms. For example, it is likely thatsome of the genes involved in human-blood diseasesaffecting specific cell types may have other functionsin Drosophila, which has a relatively simpler hemo-lymph system compared to the complexity of verte-brate blood.

These general guiding principles should not be ad-hered to dogmatically, however. For example, the genefor the metabolic disorder acute porphyria (OMIM#176000), a defect in the gene for porphobilinogendeaminase (PBG), has a clearly related gene in Dro-

Reiter et al.


sophila (E = 10�78). Although there are gene sequencesrelated to this uroporphyrinogen synthetase in manylower organisms, including yeast, the phenotype inhumans involves paralysis and seizures as a result ofsecondary neurotoxicity from the buildup of excessporphyrin precursors. The study of such secondary ef-fects of biochemical defects and the suppressors ofthese effects is more suited to an organism like Dro-sophila, which has a complex nervous system. As ithappens, the fly gene most related to PBG (CG9165)contains a P-element insertion EP(3)0419. Thus, thisgene seems to be an excellent candidate gene for studyin Drosophila.

Another limitation of the cross-genomic compari-son of human disease genes is that some of the Dro-sophila genes related to human disease genes may notbe functionally equivalent (or orthologous) to the hu-man disease gene in question, but rather may be morerelated by sequence and/or activity to another humangene that has a different function than the human dis-ease gene. It is therefore to be anticipated that theclear-hit list contains matches between human andDrosophila genes that are members of a related butfunctionally diverged gene family. True orthologs maybe identified through functional studies of individualgenes in Drosophila. Thus, an important first step inanalyzing any human disease gene in flies will be todemonstrate that the wild-type form of the human dis-ease gene can substitute for (or rescue) loss-of-functionmutants in the Drosophila gene. It is worth noting inthis regard that a significant number of human diseasegenes have very strong matches to Drosophila counter-parts (e.g., 274 disease genes = 29% match with E�10�100), suggesting that this stringent criterion offunctional equivalence will be satisfied in many cases.Our group is in the process of studying several humandisease genes using misexpression in Drosophila. Ourinitial findings indicate that at least for the humanCYP2D6 gene, the Drosophila cyp18 gene is an orthologand that regulation of the Drosophila gene can be dis-rupted via misexpression of the human gene (L. Reiter,pers. comm.).

With the above considerations and qualificationsin mind, we believe that there are broad categories ofcandidate disease genes that are likely to be particularlyamenable to study in Drosophila. Thus, among the hu-man disease, clear-hit genes, 74 result in neurologicaldisorders. Given the substantial existing evidence in-dicating that basic neuronal systems have been con-served between flies and humans, this set of diseasegenes is likely to be effectively analyzed in Drosophila.As mentioned above, analysis of Drosophila mutantsinvolved in synaptic transmission and action potentialpropagation has proven to be directly relevant to theseprocesses in vertebrates (Salzberg and Bellen 1996;Wu and Bellen 1997). Also, the 296 genes that repre-

sent developmental, neurological, cardiovascular,ophthalmologic, and hearing disorders as well as can-cers, appear to be good candidates for study using Dro-sophila because there is reason to believe that the un-derlying molecular mechanisms controlling these or-ganismic processes also are highly similar in flies andhumans.

For the individual researcher, we suggest that thebest approach to using our dataset is to query Homo-phila for a particular disease or key word representinga class of disorders. From these results, one can judgethe degree of similarity between the human and flygenes as well as the domains that are similar (for ex-ample, the HOX genes show homology only in theDNA-binding domain). The domain graphic below thebest match enables the user to determine if the bestmatch is in a known domain and not across the entiregene. One should be mindful when using this infor-mation not to discard hits with only localized domainsof homology. For example, in the case of the HOXgenes, it has been well established that functionallyorthologous genes in highly diverged species sharehigh-sequence similarity only within the DNA-bindinghomeobox domain. Yet this relatively small portion ofthe molecule seems to carry key developmental infor-mation. There are links to both OMIM for the humandisease information as well as to Flybase to determineallele and P-element information. In addition to deter-mining if the human and fly genes are likely to per-form similar functions, reasonable criteria for a good-candidate disease gene for study in Drosophila wouldinclude the following: (1) There is at least good circum-stantial evidence that the gene is involved in the dis-ease condition, (2) the mechanism by which the hu-man gene functions is poorly understood (e.g., has notbeen placed in the context of a known pathway), and,pragmatically, (3) there is at least one mutant allele inthat gene (153 genes) or a P-element insertion in ornear that gene (56 genes).

Future Development of Homophila as an InteractiveTool for Cross-Genomic AnalysisWe will continue to develop the Homophila databaseto bridge the gap between the human disease (OMIM)and Drosophila (Flybase) databases, which were notoriginally designed to facilitate cross-genomic brows-ing. Given that ∼ 4000 human disease phenotypes mayhave a genetic basis (Scriver 1995), it seems likely thatthe number of genes currently listed in OMIMwill con-tinue to grow at a rapid pace and that the frequentupdates to the Homophila database will provide re-searchers with state-of-knowledge links to Drosophilacounterparts of these genes. In addition, we currentlyare creating software to facilitate discovery of second-ary associations among human and fly genes, which isnow the focus of our next phase in development of the



database. In particular, we plan to implement a versionof the database that will allow for phenotype key wordsearches in both human and fly databases. This modi-fied alignment technology would create key wordstrings for each disease-gene entry in OMIM andeach fly-gene entry (e.g., by distilling key words fromOMIM, Flybase, Interactive Fly, or Medline reviewabstracts) and then allow researchers to search theseunordered strings against one another for statisticallysignificant similarities (e.g., neurological diseaseswith cell loss in the cerebellum). The idea would be tothen examine the fly cognates of genes responsiblefor similar diseases identified by such key wordsearches, and ask if these fly genes have some interest-ing feature or function in common (e.g., they are partof a common signaling pathway or molecular ma-chine of some kind). It also would be possible to do thisin reverse (e.g., cluster fly genes and ask if the corre-sponding human diseases share any common diseasephenotypes).

Another addition to Homophila we are planning issoftware to identify potential candidate disease genesbased on predicted phenotypes. This idea is based onthe fact that while there are many examples in whichseveral human disease genes belong to a common sig-naling pathway or functional module, there typicallyare not known diseases associated with all componentsin these pathways as defined by studies inDrosophila orother systems. In principle, one could guess the typesof disease phenotypes that might arise from mutationsin human orthologs of these other components (basedon the disease phenotypes of mutations in existingcomponents, the phenotypes of mutations of theseother components in Drosophila, and the expressionpattern of these components in mice or other verte-brates). The software we are currently developing willbe used to identify human counterparts of fly genes ina systematic fashion and to ask if any diseases match-ing the predicted phenotypes have been mapped toregions of the human genome containing those genes.We anticipate that with the input of both the humanand Drosophila genetics communities, Homophila willbecome a valuable cross-genomics tool in the post-genome-sequence era.

METHODS

Identification of Drosophila Genes Related to HumanDisease GenesThis work reflects version 3.01 of the Homophila database(released Feb. 1, 2001). Our analysis began with the OMIMmorbid map, a catalog of genetic diseases and their cytoge-netic map locations, which is available electronically at ftp://ncbi.nlm.nih.gov/repository/OMIM/morbidmap. It was notpossible to simply download the sequences related to eachdisease in the on-line version of OMIM because the protein

and nucleic sequences associated with each OMIM entry of-ten include unrelated genes mentioned in the text. Thus, amore involved procedure, relying on the NCBI Locuslink da-tabase, was required. Beginning with each of the 1792 geneticdiseases specified in the OMIM morbid map, each disease wasidentified in the Locuslink mim2loc table, which relatesOMIM entries to NCBI locus records. Each locus record thenwas used to locate the correct protein and nucleic-acid se-quence records using the Locuslink loc2UG, loc2acc, andloc2ref tables, which specify entries in the NCBI Unigene,protein, nucleic acid, and RefSeq databases, respectively. Thisprocess was simplified by downloading the Locuslink tables(mim2loc, loc2ref, loc2acc, and loc2UG) and importing themdirectly into the Homophila database. The result of this pro-cedure was a list of 4104 protein-sequence entries associatedwith 929 OMIM disease loci, and 4643 nucleic-acid sequenceentries associated with 941 OMIM disease loci. Each of theprotein-sequence entries was compared to the completeDrosophila genome sequence (Adams et al. 2000) using theBLASTPand TBLASTXprograms (Altschul et al. 1997). BLASTcomparisons were performed using BLAST v2.09 and thestandard BLOSUM 62 and E = 10 settings. Many OMIM dis-ease entries have multiple protein sequences linked to thedisease through Locuslink. The BLAST search results foreach of the probe sequences are merged, and the most sig-nificant hit (smallest E value) taken to construct the tableof clear hits (Drosophila cognates of human disease genes,Table 1).

A relational database has been implemented to allowqueries on these results and is available on-line (http://homophila.sdsc.edu) using the MySQL relational databasemanagement system (Dubois 2000). PERL scripts using theDBI package are used to convert queries entered on the Ho-mophila Web pages to SQL queries to the actual RDBMS. Acomplete list of P-element locations in the Drosophila ge-nomic sequence was kindly provided by FlyBase (Flybase1999).

ACKNOWLEDGMENTSThe authors thank the members of the human geneticscommunity for their suggestions and comments during thepreparation of this manuscript. We also thank Dr. VictorMcKusick, creator of the OMIM database that made our studypossible, for his insightful comments. This work was sup-ported in part by grants to E.B. from the NIH (NS29870 andGM60585) and NSF (IBN-9604048) and from NIH P41RR08605, National Biomedical Computation Resource whichprovides the server and also provides support to M.G. and S.C.L.T.R. was supported in part by a grant from the GlaucomaFoundation.

The publication costs of this article were defrayed in partby payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 USCsection 1734 solely to indicate this fact.

REFERENCESAdams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne, J.D.,

Amanatides, P.G., Scherer, S.E., Li, P.W., Hoskins, R.A., Galle,R.F., et al. 2000. The genome sequence of Drosophilamelanogaster. Science 287: 2185–2195.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z.,Miller, W., and Lipman, D.J. 1997. Gapped BLAST and

Reiter et al.


PSI-BLAST: A new generation of protein database searchprograms. Nucleic Acids Res. 25: 3389–3402.

Dubois, P. 2000. MySQL. New Riders Publishing, Indianapolis, IN.Flybase. 1999. The FlyBase database of the Drosophila Genome

Projects and community literature. The FlyBase Consortium.(http://flybase.bio.indiana.edu/).

Fortini, M.E. and Bonini, N.M. 2000. Modeling humanneurodegenerative diseases in Drosophila: On a wing and aprayer. Trends Genet. 16: 161–167.

Fortini, M.E., Skupski, M.P., Boguski, M.S., and Hariharan, I.K. 2000.A survey of human disease gene counterparts in the Drosophilagenome. J. Cell. Biol. 150: F23–30.

Howard, T.D., Paznekas, W.A., Green, E.D., Chiang, L.C., Ma, N.,Ortiz de Luna, R.I., Garcia Delgado, C., Gonzalez-Ramos, M.,Kline, A.D., and Jabs, E.W. 1997. Mutations in TWIST, a basichelix-loop-helix transcription factor, in Saethre-Chotzensyndrome. Nat. Genet. 15: 36–41.

Littleton, J.T. and Ganetzky, B. 2000. Ion channels and synapticorganization: Analysis of the Drosophila genome. Neuron26: 35–43.

McKusick, V.A. 2000. Online Mendelian Inheritance in Man, OMIM.(http://www.ncbi.nlm.nih.gov/omim/).

Potter, C.J.,Turenchalk, G.S., and Xu, T. 2000. Drosophila in cancerresearch: An expanding role. Trends Genet. 16: 33–39.

Reiter, L., Beir, E., and Gribskov, M. 2000. Homophila.(http://homophila.sdsc.edu).

Rong, Y.S. and Golic, K.G. 2000. Gene targeting by homologousrecombination in Drosophila. Science 288: 2013–2018.

Rubin, G.M., Yandell, M.D., Wortman, J.R., Gabor Miklos, G.L.,Nelson, C.R., Hariharan, I.K., Fortini, M.E., Li, P.W., Apweiler, R.,Fleischmann, W., et al. 2000. Comparative genomics of theeukaryotes. Science 287: 2204–2215.

Salzberg, A. and Bellen, H.J. 1996. Invertebrate versus vertebrateneurogenesis: Variations on the same theme? Dev. Genet.18: 1–10.

Scriver, C.R. 1995. The metabolic and molecular bases of inheriteddisease, 7th ed. McGraw-Hill Health Professions Division, NewYork, NY.

Wu, M.N. and Bellen, H.J. 1997. Genetic dissection of syn-aptic transmission in Drosophila. Curr. Opin. Neurobiol.7: 624–630.

Received October 25, 2000; accepted in revised form April 11, 2001



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