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Dissection of epistasis in oligogenic Bardet–BiedlsyndromeJose L. Badano1, Carmen C. Leitch1, Stephen J. Ansley1, Helen May-Simera5, Shaneka Lawson1,Richard Alan Lewis6, Philip L. Beales5, Harry C. Dietz1,2, Shannon Fisher1,3 & Nicholas Katsanis1,4

Epistatic interactions have an important role in phenotypicvariability, yet the genetic dissection of such phenomena remainschallenging1. Here we report the identification of a novel locus,MGC1203, that contributes epistatic alleles to Bardet–Biedl syn-drome (BBS), a pleiotropic, oligogenic disorder2–9. MGC1203encodes a pericentriolar protein that interacts and colocalizeswith the BBS proteins. Sequencing of two independent BBScohorts revealed a significant enrichment of a heterozygousC430T mutation in patients, and a transmission disequilibriumtest (TDT) showed strong over-transmission of this variant.Further analyses showed that the 430T allele enhances the use ofa cryptic splice acceptor site, causing the introduction of apremature termination codon (PTC) and the reduction ofsteady-state MGC1203 messenger RNA levels. Finally, recapitula-tion of the human genotypes in zebrafish shows thatmodest suppression of mgc1203 exerts an epistatic effect on thedevelopmental phenotype of BBS morphants. Our data demon-strate how the combined use of biochemical, genetic and in vivotools can facilitate the dissection of epistatic phenomena, andenhance our appreciation of the genetic basis of phenotypicvariability.

Despite major progress in elucidating the genetic basis of inheriteddisorders, much of the observed phenotypic variability cannot beexplained by mutations at a single locus, leading to the exploration ofoligogenic models of disease transmission, in which multiple lociexert a synergistic effect to modify the penetrance and/or expressivityof disease traits1.

BBS is inherited primarily as an autosomal recessive trait. How-ever, in some patients, three mutations across two BBS loci interact tomodify the onset and/or severity of the phenotype2–9. To date, eightBBS genes (BBS1–BBS8) have been identified2,5,9–16, and a combi-nation of in vivo and in vitro evidence suggests that BBS is a disorderof basal bodies and cilia10,17.

The degree of clinical variability in BBS is not fully reconciled byinteractions between the known BBS genes18. We reasoned that lociencoding proteins pertinent to the BBS functional circuit would bestrong candidates to contribute modifying alleles. To identify suchloci, we performed multiple rounds of yeast two-hybrid screens andidentified .60 putative interactors. However, comparison of thesewith our recently described ciliary proteome9 revealed that only asingle sequence, MGC1203 (also known as CCDC28B; GenBankaccession number NM_024296), was present in both data sets. Thiscomputationally predicted polypeptide is composed of 241 aminoacids, bears no recognizable motifs, and interacts with BBS4 in yeast(Supplementary Fig. 1a).

To investigate this interaction, we expressed epitope-taggedMGC1203 and BBS4 in mammalian cells. Immunoprecipitations

with BBS4 followed by immunoblotting for MGC1203 yielded asingle band of the predicted size of 35 kDa (Fig. 1a). This interactionwas not restricted to BBS4 but was seen for every BBS protein tested(Fig. 1a).

Next, we raised a polyclonal antibody against MGC1203 (Sup-plementary Fig. 1b) and localized MGC1203 near centrosomes andbasal bodies of HeLa or IMCD3 cells, a localization pattern identicalto BBS4, BBS6 and BBS810,19,20 (Fig. 1b). We also found MGC1203to be spatially coincident with the BBS proteins in tissues pertinentto the disorder, including retina, pericardium and limb epithelium(Fig. 1c; Supplementary Fig. 1c).

Our data suggested that MGC1203 might be relevant to the geneticaetiology of BBS. To test this hypothesis, we confirmed its genomicstructure (Supplementary Fig. 2) and screened 226 unrelated BBSpatients without preselecting for mutational load in the known BBSloci. In an initial BBS cohort and ethnically matched controls, wefound a C ! T transversion at the penultimate position of exon 3(C430T) present in the heterozygous state in 3/64 unrelated BBSpatients, compared to 4/274 controls. Analysis of a second cohortshowed an even greater enrichment, with 11/162 unrelated patientscarrying the 430T allele. Overall, the 430T variant of MGC1203 waspresent in 6.2% of BBS patients compared to 1.4% of controls,showing significant association with BBS (Fisher’s exact testP , 0.006). As a second, independent, test, we screened MGC1203in all available parents of the BBS patients analysed. We identified 27trios with a 430T heterozygous parent, and performed TDT analysis.We found the 430T allele transmitted to patients in 20/27 trios,deviating significantly from the expected 50:50 distribution(P , 0.007).

The MGC1203 mutations are probably insufficient to cause BBS.Not only did we find no patients with homozygous or compoundheterozygous MGC1203 mutations, but we found one unaffected430T homozygous parent. Moreover, five patients carried twomutations at a known BBS locus (Supplementary Table 1), suggestingthat the observed association between the 430T allele and BBS mightreflect an epistatic relationship. In three of the 14 families withthe 430T allele, some, but not all, affected individuals inherited theMGC1203 mutation (Supplementary Table 1). In each case, theunbiased clinical view (blinded to the genotype) was that 430T-bearing individuals were more severely affected. In family AR46,individual 204 (BBS1: Y113X/M390R; MGC1203: 430C/C) was firstdiagnosed with retinitis pigmentosa (RP) at the age of 12, whereassibling 205 (BBS1: Y113X/M390R; MGC1203: 430C/T) presentedwith aggressive RP at age five, ataxia and gastroschisis. In familyAR709, patient 203 (BBS1: M390R/E549X; MGC1203: 430C/C)developed RP at 12 years of age, whereas 204 (BBS1: M390R/E549X; MGC1203: 430C/T) was diagnosed with RP at six years of

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1McKusick-Nathans Institute of Genetic Medicine, 2Howard Hughes Medical Institute, 3Department of Cell Biology, 4Wilmer Eye Institute, Johns Hopkins University, Baltimore,Maryland 21205, USA. 5Molecular Medicine Unit, Institute of Child Health, University College London, London WC1N 1EH, UK. 6Departments of Molecular and Human Genetics,Ophthalmology, Pediatrics, and Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.

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age, had 20/400 vision and underwent surgery at infancy forHirschsprung disease. Finally, in family AR151, although bothpatients were diagnosed with RP at similar ages (six and nine),individual 204 (MGC1203: 430C/T) presented with a more aggres-sive form of the disease with macular involvement, as well as asthma,profound speech delay, and thoracolumbar scoliosis. A fourth sib-ship, PB029, provided a more definitive phenotypic stratification. In

this family, both affected individuals are homozygous for thecommon M390R mutation in BBS1 and are C430T heterozygotes.However, the unaffected father is also M390R homozygous, andthis family has been put forth as a possible example of complexinheritance4 (Fig. 2a). The patients inherited the MGC1203 430Tallele from their mother, whereas the father is homozygous 430C(Fig. 2b).

The 430T allele might be causally related to BBS or be in linkagedisequilibrium with another mutation. Although this variant resultsin a silent change, its position in the penultimate base of exon 3 raisedthe possibility that it might affect splicing. To investigate this, wesuppressed nonsense-mediated decay (NMD) with emetine in celllines from two unrelated 430T heterozygous patients and sequencedMGC1203. Although we did not detect aberrant splicing at the 3 0

junction of exon 3, we found that, in addition to all normal spliceisoforms of MGC1203, 10% of the sequenced products contained a5-bp deletion in the acceptor site at the 5

0junction of the same exon.

This deletion was generated by the inappropriate utilization of acryptic splice acceptor site (Fig. 2c) and resulted in a PTC. Analysiswith ESEfinder21 predicted that the 430T variant might improve anexonic splice enhancer (ESE) motif recognized by the SR proteinSC35 (for splicing component of 35 kDa)22 (Fig. 2c). Such proteinsstrengthen the binding of the spliceosome to suboptimal, upstreamacceptor binding sites. To test this possibility, we performed real-timepolymerase chain reaction with reverse transcription (RT–PCR) oncell lines with either the 430C/C or 430C/T genotype, with or withoutemetine. We found that the steady-state abundance of MGC1203message was greater in all emetine-treated cells, suggesting that NMDoccurs irrespective of genotype. However, we found a reduction of,20% in MGC1203 mRNA levels in untreated 430C/T cells (Fig. 2d),suggesting that a larger fraction of mRNA from the 430C/T genotypecontains a PTC.

We next constructed a series of MGC1203 minigenes (spanningfrom exon 2 to exon 4) and monitored the relative transcription ofthe mis-spliced message (del) relative to total MGC1203 transcrip-tion. The 430T allele showed a marked increase in del mRNAproduction compared to a 430C minigene from both a normalindividual and a 430C/T heterozygous patient (Fig. 2e). Notably,reversion of the 430Tresidue to 430C restored the relative productionof the del allele to wild-type ratios (Fig. 2e). Finally, we suppressedSC35 and ASF/SF2 (alternate splicing factor/splicing factor 2) mRNAwith previously reported siRNA oligomers23,24 and analysed theabsolute amounts of del MGC1203 transcript. In agreement withour earlier data, we found minimal production of the mis-splicedtranscript from either the 430C haplotype or the mutagenized430T ! C haplotype (2 and 8 pg, respectively) compared with170 pg produced from the 430T minigene (Fig. 2f). Suppression ofapproximately 70% of SC35 or.90% ofASF/SF2message resulted ina dramatic reduction of the deleted MGC1203 species to 70 pg and3 pg, respectively, suggesting that the 430T allele enhances the effectof SR proteins to potentiate the cryptic splice site (Fig. 2f).

Cumulatively, our data suggest that a hypomorphic MGC1203mutation in humans can exert an epistatic effect on BBS mutations.To investigate this possibility in vivo, we undertook an antisensemorpholino strategy in zebrafish. We first established that eachtranscript is expressed both maternally and zygotically, and isfound along the rostrocaudal axis of the embryo (SupplementaryFig. 3). We designed morpholinos against the single BBS4 and BBS6orthologues, the two most extensively characterized BBS genes19,20,and the single MGC1203 orthologue. Although we did not detectappreciable numbers of late embryonic phenotypes, such as renal orheart positioning/looping defects, bbs4 morphants showed dosage-dependent phenotypes during somitogenesis (Fig. 3; SupplementaryFig. 4). Severely affected embryos showed dorsal thinning andshorter body length. By the 12-somite stage, the notochord waskinked and sometimes twisted, accompanied by widening of thesomites. Finally, we observed cell detachment along the neural tube,

Figure 1 | MGC1203 interacts and colocalizes with BBS proteins.a, HA–MGC1203 immunoprecipitates with all BBS proteins tested (toppanel). Cell lysates probed with the anti-HA antibody (middle panel) andimmunoprecipitates probed with an anti-Myc monoclonal antibody(bottom panel) are shown as controls. MW, molecular weight; IP,immunoprecipitation with the indicated antibody; Probed, western blotwith the indicated antibody. b, In IMCD3 cells, MGC1203 (green)colocalizes with g-tubulin (red). DNA was stained with DAPI (4,6-diamidino-2-phenylindole). c, MGC1203 colocalizes with BBS1 and BBS4 intissues. In the adult mouse retina, all proteins show overlapping patterns ofexpression (arrow). Similarly, all proteins are expressed ubiquitously in thepericardium of the developing heart and the epithelium surrounding thedigits of embryonic day (E) 15.5 embryos. OS, outer segment; CC,connecting cilium; IS, inner segment; ONL, outer nuclear layer; INL, innernuclear layer; GCL, ganglion cell layer.

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most frequently in the head and tail regions—reminiscent of defectsin neurulation and neural cell adhesion25 (Fig. 3) and consistent withthe recently demonstrated genetic interaction of some basal bodyproteins with the planar cell polarity pathway26,27. These phenotypescould be rescued by co-injection of 50 pg of bbs4 RNA, which by itselfdid not show any remarkable effect. We observed similar phenotypeswith a bbs6 translational-blocking morpholino, although the severityand incidence of the phenotypes was not as prominent (Supplemen-tary Fig. 4, 5). We then injected a splice-blocking morpholino againstmgc1203 and observed phenotypes that overlapped the bbs4 and bbs6defects. Mgc1203 morphant embryos had shorter body axes butdid not display appreciable dorsal thinning. They also showedmoderately widened, but noticeably kinked, notochords. Somiteswere not widened, but were shaped abnormally and lacked defi-nition. Finally, we observed cell detachment along the neural tube,most prominently in the head region (Fig. 3; Supplementary Fig. 4).These phenotypes correlated with the relative potency of eachmorpholino; mild morphants showed ,60% reduction of mgc1203message, whereas severe morphants had .90% reduction (Sup-plementary Fig. 6). These phenotypes were also specific to themorpholino as they could be rescued by co-injection of 75 pg ofmgc1203 RNA, which by itself does not produce any noticeableeffects. In addition, injection of a second mgc1203 morpholino thatbinds to the 5 0 untranslated region of mgc1203 produced comparablephenotypes (data not shown).

We next performed double morpholino injections. Injection ofeither 5 ng of mgc1203 morpholino or 3 ng of bbs4 morpholinoconsistently produced .80% phenotypically normal embryos; wheninjected together, however, 68% of embryos had either the moderateor severe phenotypic features of a higher dose of bbs4 morpholinoalone (Fig. 3; Supplementary Fig. 7a). Notably, co-injection of75 pg of mgc1203 RNA with the two morpholinos rescued thedouble-morphant phenotype, further suggesting that the synthetic

phenotype was due to the interaction between the morpholinos, asopposed to potentially non-specific morpholino effects.

The interaction between MGC1203 and BBS6 was morepronounced. A subeffective dose of mgc1203 morpholino enhancedthe phenotypes caused by low doses of bbs6 morpholino (Sup-plementary Fig. 7b), producing a higher incidence of BBS defectsin conjunction with a low dose of bbs6 morpholino than seen in anyexperiment with a higher dose of bbs6 morpholino alone. Signifi-cantly, partial depletion of both mgc1203 and either bbs4 or bbs6produced phenotypes not found in any mgc1203 single morphants,such as dorsal thinning and broadening of the somites.

Finally, we tested for interaction of mgc1203 with bbs1. The bbs1morphant phenotypes produced with two non-overlappingtranslational-blocking morpholinos were indistinguishable frombbs4 or bbs6 morphants, although the number of affected embryoswas modest (Fig. 4a, b). However, double bbs1;mgc1203 morphantsshowed phenotypes that were not only reminiscent of bbs4;mgc1203and bbs6;mgc1203 double injections, but were the most severe withrespect to the disruption of somitic definition (Fig. 4a, b).

Our studies indicate that MGC1203, a protein identified through adouble filter of yeast two-hybrid and computational determinationof ciliary proteins, is involved in the pathogenesis of BBS bycontributing hypomorphic mutations to an already sensitized gen-etic background. The 430T mutation exerts its effect at the RNA levelby enhancing the use of a cryptic splice junction in a suboptimalcontext that is probably stabilized by SR proteins such as SC35 andASF/SF2. Although there are examples in the literature of ESEmutations, the effect of such changes is typically the inefficientinclusion/exclusion of an alternatively spliced exon21, whereas herethis phenomenon leads to the partial loss of MGC1203 message. Thismodel is reminiscent of erythropoietic protoporphyria, where pene-trance is enhanced by the loss of a modest amount of ferrochelatase(FECH) mRNA owing to aberrant splicing and NMD28, although in

Figure 2 | Epistasis and splice defects of the 430T variant. a, b, In pedigreePB029, the presence of the MGC1203 430Tallele segregates with a penetrantphenotype, as the unaffected father is homozygous mutant for BBS1 (a) butwild type (WT) for MGC1203 (b). c, In addition to wild-type MGC1203, amutant transcript is also produced, containing a 5-bp deletion (orange box).The position of the C430T change is shown in the context of a putativebinding site for SC35 (purple box; the consensus sequence matrix is shownunderneath); the real-time PCR probe that detects the RNA deletion isindicated (Del probe). d, Real-time RT–PCR results with a wild-type (WT)probe with or without emetine in lymphoblastoid cells from 430C/C and

430C/T individuals. The cycles needed to reach a determined threshold areused to measure template abundance, normalized to 18S RNA. e, Theabundance of MGC1203 del/WT mRNA species is measured by thedifference in PCR cycles between the two assays. Mutagenizing the T allelebase back to a C restores the del/WT balance observed in the 430C minigenesfrom both backgrounds (14-C and 316-C). f, Quantification of del amountsusing a plasmid standard curve yielded comparable results; suppression ofSC35 or ASF/SF2 significantly reduces the amount of del mRNA.Experiments were performed in triplicate; Error bars are s.e.m.

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our example, the penetrance modification is contributed by an alleleat a discrete, yet functionally related, locus.

Establishing the effect of epistatic variants can be challenging,given that hypomorphic mutations are typically in Hardy–Weinbergequilibrium in the population. Our studies highlight the usefulnessof combinatorial approaches for the dissection of such phenomena

and exemplify how human genetics can lead to the functionalinvestigation of phenotypic synthesis and further validation ofpreviously established theoretical models29,30. Improved understand-ing of the relationships between the components of a given molecularcircuitry will be important for improving the ability of genotypicinformation to predict the phenotype in both oligogenic andcomplex traits.

METHODSYeast two-hybrid assay and immunoprecipitations. We performed RT–PCR onhuman RNA to amplify the open reading frame of BBS-1, -2, -4, -5, -6, -7 and -8,which were then cloned into the pSOS vector (Stratagene) and pCMV–Myc andpCMV–HA vectors (Clontech). To identify putative BBS4 interacting proteins,we used pSOS-BBS4 as bait in the Cytotrap yeast two-hybrid assay (Stratagene)with a fetal brain library as prey as described19. To confirm putative interactors,we assessed their ability to bind the BBS proteins by co-immunoprecipitation(co-IP) in an in vitro mammalian system as described10 (SupplementaryMethods).Mammalian cell culture and microscopy. HEK293T, HeLa and IMCD3 cellsused for transient transfection, co-immunoprecipitations and immunohisto-chemistry were grown in standard conditions (Supplementary Methods). Forfluorescence microscopy, cells grown on glass coverslips were treated asdescribed19 (Supplementary Methods).

Figure 3 | Genetic interaction of mgc1203 with bbs4 in zebrafish. Liveembryos showing characteristic class I (mild) or class II (severe) phenotypesof animals injected either with a bbs4 morpholino alone, a high dose ofmgc1203 morpholino, or injected with low dose of both morpholinos. Sideviews (left panels) illustrate the shortening of the body axis and, for bbs4morphants, dorsal thinning. Note the detachment of cells along therostrocaudal axis. Dorsal views (middle panels; magnified in right panels)show somitic and notochordal defects in the morphants. Originalmagnification: £6.6 (left and middle panels); £26.4 (right panels).

Figure 4 | Potent interaction between mgc1203 and bbs1. a, Live zebrafishimages showing a range of embryo phenotypes for the bbs1 morpholinoeither alone, or in combination with the mgc1203 morpholino. Note theparticularly poor somitic definition of bbs1;mgc1203 double morphants.b, Plot of the prevalence of phenotypes arising from the various injections.Original magnification: £6.6 (top and middle panels); £26.4 (bottom panels).

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BBS patients and mutational analyses. Subject examination, clinical data, andsamples were obtained under informed consent, and a diagnosis of BBS wassecured according to established criteria. To sequence the complete open readingframe and exon–intron boundaries of MGC1203, we designed PCR primers asdescribed12 (Supplementary Methods). Primer sequences are available uponrequest. To analyse the resulting sequence reads, we used the Sequenchersequence alignment program (Gene Codes).MGC1203minigene construction and analysis. We amplified a 2.5-kb genomicfragment from exon 2 to exon 4 of MGC1203 by PCR with TaKaRa LA Taq DNApolymerase and cloned it into the pcDNA3.1(þ) vector (Invitrogen). Allconstructs were sequenced verified across their entire length by bidirectionaldye-primer sequencing. Transfections were carried out in HEK293T cells withthe Calcium Phosphate kit (Invitrogen) as described above, and cells werecollected 24 h later. Total RNA was extracted and first-strand cDNA was preparedas described earlier. First-strand cDNA was then used as template in quantitativereal-time PCR assays. Assays for the MGC1203 transcript and the 5-bp deletedproduct, as well as 18S RNA and neomycin, were custom made by ABI usingFAM-MGB probes (Applied Biosystems). Real-time PCR reactions were carriedout using a 7900HT Sequence Detection System (Applied Biosystems).Morpholinos and embryo manipulations. Translational and splice morpholi-nos against bbs4, bbs6 and mgc1203, as well as a control morpholino, weredesigned by and obtained from Gene Tools (see the Supplementary Methods).One nanolitre of diluted morpholino was injected into wild-type zebrafishembryos at the 1-to-2-cell stage. Injected embryos were observed for 24–30 h andscored. For RNA rescue experiments, bbs4, bbs6 and mgc1203 mRNA wastranscribed in vitro using the SP6 mMessage mMachine kit (Ambion). Mor-phant embryos were classified into two graded phenotypes depending on therelative severity compared to age-matched controls from the same clutch (see theSupplementary Methods for a description).

Received 4 August; accepted 25 October 2005.Published online 4 December 2005.

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank the BBS patients and their families for theircontinued support and encouragement. We also thank J. Gerdes andA. McCallion for their thoughtful comments on the manuscript. This work wassupported by grants from the National Institute of Child Health andDevelopment (N.K.), the National Institute of Diabetes, Digestive and Kidneydisorders (N.K.), the National Institute for Arthritis and Musculoskeletaldisorders (S.F.), the Polycystic Kidney Disease Foundation (J.L.B. and N.K.), andthe Medical Research Council (P.L.B.). R.A.L. is a Senior Scientific Investigator ofResearch to Prevent Blindness. P.L.B. is a Senior Wellcome Trust Fellow. H.C.D.is an Investigator of the Howard Hughes Medical Institute.

Author Information Reprints and permissions information is available atnpg.nature.com/reprintsandpermissions. The authors declare no competingfinancial interests. Correspondence and requests for materials should beaddressed to N.K. (katsanis@jhmi.edu).

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