Incomplete penetrance and phenotypic variability characterize Gdf6-attributable oculo-skeletal

Incomplete penetrance and phenotypic variabilitycharacterize Gdf6-attributable oculo-skeletalphenotypes

Mika Asai-Coakwell1, Curtis R. French2, Ming Ye1, Kamal Garcha3, Karin Bigot4,

Anoja G. Perera5, Karen Staehling-Hampton5, Silvina C. Mema1, Bhaskar Chanda1,

Arcady Mushegian5, Steven Bamforth6, Michael R. Doschak7, Guang Li7, Matthew B. Dobbs8,

Philip F. Giampietro9, Brian P. Brooks10, Perumalsamy Vijayalakshmi11, Yves Sauve1,

Marc Abitbol4, Periasamy Sundaresan12, Veronica van Heyningen13, Olivier Pourquie5,

T. Michael Underhill3, Andrew J. Waskiewicz2 and Ordan J. Lehmann1,6,�

1Department of Ophthalmology, University of Alberta, Edmonton T6G 2H7, Canada, 2Department of Biological

Sciences, University of Alberta, Edmonton T6G 2E9, Canada, 3Department of Cell and Developmental Biology,

University of British Columbia, Vancouver V6T 1Z3, Canada, 4CERTO–EA No 2502 du ministere de la recherche,

Faculty of Medicine 75015, Paris, France, 5Stowers Institute for Medical Research, Kansas, MO 64110, USA,6Department of Medical Genetics, University of Alberta, Edmonton T6G 2H7, Canada, 7Department of Pharmacy and

Pharmaceutical Science, University of Alberta, Edmonton, Canada, 8Department of Orthopedic Surgery, Washington

University, St Louis, MO 63130, USA, 9Department of Medical Genetic Services, Marshfield Clinic, Marshfield,

WI 54449, USA, 10Ophthalmic Genetics and Visual Function Branch, NEI, NIH, Bethesda, MD 20892, USA,11Department of Paediatric Ophthalmology and Strabismus, Aravind Eye Hospital, Madurai, Tamilnadu, India,12Department of Genetics, Aravind Medical Research Foundation, Madurai, Tamilnadu, India and 13MRC Human

Genetics Unit, Edinburgh EH4 2XU, UK

Received October 8, 2008; Revised and Accepted December 19, 2008

Proteins of the bone morphogenetic protein (BMP) family are known to have a role in ocular and skeletaldevelopment; however, because of their widespread expression and functional redundancy, less progresshas been made identifying the roles of individual BMPs in human disease. We identified seven heterozygousmutations in growth differentiation factor 6 (GDF6), a member of the BMP family, in patients with both ocularand vertebral anomalies, characterized their effects with a SOX9-reporter assay and western analysis, anddemonstrated comparable phenotypes in model organisms with reduced Gdf6 function. We observed a spec-trum of ocular and skeletal anomalies in morphant zebrafish, the latter encompassing defective tail formationand altered expression of somite markers noggin1 and noggin2. Gdf61/2 mice exhibited variable ocularphenotypes compatible with phenotypes observed in patients and zebrafish. Key differences evidentbetween patients and animal models included pleiotropic effects, variable expressivity and incompletepenetrance. These data establish the important role of this determinant in ocular and vertebral development,demonstrate the complex genetic inheritance of these phenotypes, and further understanding of BMPfunction and its contributions to human disease.

�To whom correspondence should be addressed. Tel: þ1 780 492 8550; Fax: þ1 780 492 6934; Email: [email protected]

# The Author 2009. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2009, Vol. 18, No. 6 1110–1121doi:10.1093/hmg/ddp008Advance Access published on January 6, 2009

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INTRODUCTION

The Growth Differentiation Factors (GDFs) are membersof the bone morphogenetic proteins (BMP) sub-family oftransforming growth factor-beta (TGF-b) signaling ligands,known to regulate patterning during development (1). Wepreviously demonstrated GDF6’s role in ocular developmentby characterizing a GDF6-encompassing copy numbervariant in a patient with ocular colobomata, and recapitulatingthe human phenotype in gdf6a morphant zebrafish (2). Suchresults, combined with comparable findings in Xenopus (3),suggested that GDF6 mutations may underlie a rangeof ocular phenotypes, including one or more components ofthe microphthalmia, anophthalmia or coloboma (MAC)developmental spectrum. In addition, in keeping withthe broad and incompletely defined roles of BMPs, it wasprobable that GDF6 mutations contributed to other humandisorders.

The BMPs were originally identified through their capacityto induce bone formation (4,5) and are now recognized to havecritical roles patterning a diverse range of tissues includingbone, heart, lungs and kidney (6–10). Over the last 10 yearssignificant progress characterizing the phenotypes resultingfrom mutation of BMPs or their receptors has ascribedhuman disease phenotypes to one-quarter of the BMP family(Online Mendelian Inheritance of Man, http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim), predominantly type I or typeII BMP receptors [ACVR1 (11), ACVRL1 (12), BMPR1A(13), TGFBR1 (14), BMPR1B (15), BMPRII (16)] ratherthan signaling ligands [BMP4 (17), BMP15 (18,19), GDF5(20–24)]. This distribution likely reflects the challenges dis-cerning human disease phenotypes for ligands that exhibitfunctional redundancy (25). The contrast between the limitedhuman phenotypes of individual ligands compared with thebroad expression pattern and expansive range of phenotypesassociated with BMP loss in model organisms implies thatonly a fraction of the human phenotypes attributable to eachhave been identified. This is clearly illustrated by the datafrom BMP4, where human mutations are primarily associatedwith microphthalmia (17), while murine loss of functioncauses defects in multiple systems: cardiovascular (26,27),craniofacial (28), ocular (25,29), reproductive, limbs, digits(30) and auditory (31). Increasing the proportion of BMPswith a defined function is thus of dual clinical and scientificimportance—advancing understanding of the molecular basisof human disease, and in view of the disparate early andlate-onset functions of individual BMPs (20,23,24,32–34),offering potential to inform understanding of multipleaspects of human genetics.

Accordingly, we set out to define GDF6’s function ingreater detail and present data demonstrating a broad role inhuman disease. At the outset of our studies, two lines of evi-dence indicated that, like other BMPs, GDF6 at a minimumpossessed an appreciable skeletal developmental role. Thefirst was data from a murine model demonstrating Gdf6’sregulation of murine skull and carpal/tarsal joint formation(35), while the second, linkage mapping of two skeletal dis-orders [Klippel-Feil (KF) syndrome (36) and Split hand andfoot malformation (SHFM) (37)] to the vicinity of GDF6(Fig. 1A), was also compatible with a skeletal function. KF

syndrome, characterized by defective cervical, thoracic orlumbar vertebral segmentation (38), is frequently associatedwith other skeletal (scoliosis, rib abnormalities, Sprengel’sdeformity) and non-skeletal (deafness, cardiovascular, ocularand renal) anomalies (39–41). Some of these (e.g. deafnessand rib anomalies) correlate with the extensive expressionpattern of Gdf6 (35). Furthermore, existence of a KFsubtype with an ocular phenotype [Wildervanck syndrome(42)] is compatible with the possibility that GDF6 causesboth ocular and skeletal disease. The mapping of the sixthSHFM (43) locus to a large GDF6-encompassing interval(37) raised the possibility that this gene might underlie thisskeletal phenotype.

In light of these factors supporting GDF6 involvement inhuman ocular and skeletal disorders, we screened patientswith ocular and skeletal developmental anomalies for GDF6mutations. We investigated the biological significance of arepresentative subset of the mutations identified with a repor-ter gene assay and western blot analysis, and characterized theeffects of perturbed Gdf6 function in two model organisms(murine and zebrafish). Our results indicate that GDF6mutations result in variable ocular and skeletal phenotypeswith evidence of non-Mendelian inheritance, accordingclosely with comparable features in animal models ofdecreased Gdf6 function. Such findings have implicationsfor deciphering interactions between members of the BMPfamily and provide a model for studying the contribution ofcomplex inheritance patterns to human disease (44,45).

RESULTS

Sequencing and mutation analysis

DNA samples from 489 patients with ocular anomalies(micropththalmia, anophthalmia and coloboma), and 81patients with vertebral segmentation anomalies were screenedfor GDF6 mutations by sequencing amplicons encompassingthe two exons and splice sites. A subset of 32 samples werescreened for copy number alteration by real-time quantitativePCR (qPCR) (TaqManw), yielding normal results. Hybridiz-ation of a DNA sample from the proband of the8q21.11-q22.3 linked SHFM pedigree to a CGH array with amean 6 kb oligonucleotide probe spacing (46), identified nocopy number variations in the 3 Mb region encompassingGDF6.

Sequencing identified two heterozygous sequence changesin exon 1 (125 g!t; 356a!g) and five heterozygouschanges (647 g!a, 746c!a, 758a!t, 980c!a and1271a!g) in exon 2 of GDF6. These sequence alterations,which were absent from dbSNP (http://www.ncbi.nlm.nih.gov/SNP/index.html) and a minimum of 366 control chromo-somes (Table 1), result in the following amino acid alterations:Gly42Val (G42V), Gln119Arg (Q119R), Asp216Gly (D216G),Ala249Glu (A249E), Gln253Leu (Q253L), Pro327His (P327H)and Lys424Arg (K424R) (Fig. 1B). Four amino acid alterations(Q119R, D216G, Q253L and P32H) were associated withocular phenotypes, two with skeletal phenotypes (G42V andK424R), while one alteration (A249E) identified in three pro-bands, was associated with either ocular or skeletal phenotypes(Table 1).

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Figure 1. Mutational analysis of GDF6. (A) Schematic representation of skeletal and ocular phenotypes mapping to chromosome 8q21-8q23. (B) Domain struc-ture of GDF6 transcript comprising: signaling ligand (light grey), putative propeptide (dark grey) and TGF-domain (black) with positions of missense mutations.Below, alignment (Clustal W) of selected amino acids encompassing regions altered in patients with ocular (o), skeletal (s) or ocular and skeletal (os) phenotypes(Mut). Note human (Hum) sequences compared with paralogs [bovine (Bov), mouse (Mus), rat (Rat), Xenopus (Xen), Danio (Dan)] and orthologs GDF5 andGDF7. Photograph of unilateral microphthalmia (C and D) in an individual with heterozygous GDF6 mutation P327H, and corresponding fundus photographs (Eand F). (G) In three pedigrees a phenotypically unaffected parent (M1, F2 and M3) carries the mutation as illustrated by enzyme digestion (A249E, P327H, andQ253L) and gel separation [P (proband), F (father), M (mother), C (control), L (Ladder)]. (H) Subset of chromosome 8q22 microsatellite markers used forhaplotype analysis of three patients carrying A249E and (I) the four informative microsatellite markers demonstrating Klippel-Feil proband 3, is unrelated tocoloboma proband 1 and microphthalmia proband 2.

Table 1. Summary of mutations identified in GDF6

Proband Mutation Phenotype Penetrance Enzyme (no. of controls)Ocular Skeletal

1 A249E Coloboma Post-axial polydactyly Incomplete BsrBI (0/646)2 A249E Microphthalmia None – BsrBI (0/646)3 A249E None Klippel-Feil Full (50) BsrBI (0/646)4 K424R None Hemi-vertebrae – Tsp509I (0/646)

Rib malformation5 Q253L Microphthalmia None Incomplete HpyCH4V (0/366)6 P327H Microphthalmia None Incomplete HpyCH4V (0/366)7 Q119R Microphthalmia None – BsrI (0/366)8 G42V None Spondylothoracic dysostosis – SphI (0/366)9 D216G Microphthalmia None – TaqaI (0/366)

‘–’ Denotes unknown because of the nature of DNA collection from which samples were derived.

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The seven residues affected vary in their degree of conser-vation, with two conserved in all vertebrates (K424R andD216G), four conserved in mammals (G42V, Q119R,Q253L, and P327H), while A249E is the least conserved(Fig. 1B). Derivation of the DNA samples from historical col-lections, some established more than a decade ago, limited theability to recall family members. However, for three of theseven amino acid alterations additional DNA samples couldbe obtained, allowing segregation to be assessed. In each ofthese cases, A249E (proband 1), Q253L (proband 5) andP327H (proband 6), the presence of the alteration in an unaf-fected parent indicates incomplete penetrance (Fig. 1G).Sequence and structure analyses demonstrate that K424Raffects the mature TGF-b domain that is invariant in vertebrates(Fig. 1B) and maps to the convex surface of the cystine knot,which is the region of interaction with ligands and solubleinhibitors. Activin A, another member of the BMP superfamily,interacts with the minimal inhibitory module of follistatinthrough multiple contacts mapped to this surface, withcharged residues in both molecules forming an extensivenetwork of hydrogen bonds (47). Replacement of this lysinewith a larger arginine residue increases the similarity ofGDF6 to GDF5 and GDF7 (members of the same clade asGDF6) that have an arginine in this position, and may poten-tially decrease the specificity of the interaction with GDF6’sreceptor(s). Similarly, the residue altered by D216G is invariantin 15 of the 19 other BMPs [including GDF5 and GDF7(Fig. 1B), as well as GDF 8, 9, 11; BMP 2, 4–8, 10, 15 (Clus-talW alignment, data not shown)]. Of the mutations identifiedin this study, the A249E alteration affects the least conservedresidue and is located in the TGF-b prodomain, a regionthought to facilitate correct folding of the mature secretedpeptide (48). A249E was identified in three probands fromgeographically disparate countries [Table 1; (49,50)] andgenotyping with eight microsatellites and six single nucleotidepolymorphism (SNPs) of which four were informative(Fig. 1H), demonstrated that at least two of these individualscarry different haplotypes (Fig. 1I).

Reporter gene and protein expression assays

To characterize the functional consequences of the amino acidchanges, two alterations (A249E and K424R) were selectedthat lie in different GDF6 domains (Fig. 1B). A functionalassay employing a SOX9-responsive reporter was used toevaluate BMP signaling. SOX9 plays a central role in chondro-genesis, and as its expression/activity is exquisitely sensitiveto changes in BMP/GDF activity, this reporter gene providesa reliable read-out on the status of BMP/GDF signaling(51,52). Accordingly, to assess chondrogenic potential,expression constructs for GDF6 and mutants A249E andK424R were co-transfected with a SOX9-responsive reportergene into primary limb mesenchymal (PLM) cells and theactivities of each determined in replicate experiments. Con-sistent with the role of GDF6 in skeletal development (35),expression of wild-type GDF6 led to 3.4-fold increasedSOX9 reporter activity. In contrast, expression of GDF6–A249E and GDF6–K424R constructs resulted in 2.9-foldand 2.4-fold increases in reporter gene activity (P , 0.034and P , 0.002; t-test) (Fig. 2A). Such diminished activation

of the reporter by the mutant GDF6 constructs provides evi-dence that these mutations alter GDF6 function.

Western blot analysis of the whole cell lysates and mediafrom wild-type and mutant GDF6 was next undertaken todetermine the effects of the two representative mutations onprotein expression and secretion. GDF6 cDNA was mutagen-ized for the two mutations (A249E and K424R), tagged with aV5 epitope, and transiently transfected into COS7 cells. Pro-teins collected from the media and whole cell lysates wereseparated by sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis, transferred to membranes and probed with V5antibody. The western blot revealed the presence ofpro-GDF6 (55 kDa) in both media and cell lysates, with themature GDF6 ligand (approximately 16 kDa) present only inthe media as a doublet (Fig. 2B). Although neither mutantconstructs exhibited abnormal formation of pro-GDF6 normature GDF6 protein, the level of secreted mature GDF6was reduced with A249E (23%) and K424R (83%) mutantscompared with wild-type [ImageJ (53)] (Fig. 2B). Inclusion ofa-tubulin (whole cell lysate) and secreted alkaline phosphatase

Figure 2. Functional analyses of GDF6 mutants. (A) Significant differences inactivity of A249E and K424R-containing mutants in the SOX9-reporter luci-ferase assay were observed, compared with wild-type. [Reporter gene activityexpressed in relative light units, normalized to the control; the percentagereduction compared with wild-type are: A249E ¼ 14% (0.1098), K424R ¼30% (0.0897); wild-type ¼ 0.1273]. (B) Western blot analysis of wild-typeGDF6 and mutants A249E and K424R. Both pro-GDF6 (55 kDa) andmature GDF6 (15 kDa) were present in the supernatant (lanes 1–3), withpro-GDF6 detected in the cell lysates (lanes 4–6). Reduced amounts ofmature GDF6 were observed in mutant GDF6 (lanes 2 and 3) when comparedwith wild-type (lane 1). Secreted alkaline phosphatase and a-tubulin controlsdemonstrate proper translation and secretion of control protein in supernatantand cell lysates, respectively. (UN ¼ untransfected control).


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(supernatants) provided controls for consistent loading andsecretion (Fig. 2B). Co-transfection of the GDF6–V5 con-structs with b-galactosidase enzyme construct and subsequentassay for b-galactosidase activity (Promega) revealed equaltranscription efficiency between wild-type and mutantV5-tagged GDF6 (data not shown).

Zebrafish model

In light of the variable human phenotypes observed withGDF6 mutations (Fig. 1C–F, Table 1) and the incompletepenetrance evident with three of seven mutations (Fig. 1G),we studied a gdf6a zebrafish model to determine if comparablefeatures were present. gdf6a function was inhibited using mor-pholino antisense oligonucleotides (MO). The first (gdf6aMO1)targets the 50-splice site, while the second (gdf6aMO2) targetsthe intron 1–exon 2 boundary (2). One cell zebrafishembryos were injected with gdf6aMO1 or gdf6aMO2 at a con-centration of 5–10 mg/ml (2,54) together with 2.5 mg/ml tar-geting the translation start site of p53 (p53MO) (55) to reduceapoptotic cell death (56). The prevalence of skeletal anomalies(curled or kinked tails) was much lower than that of ocularanomalies (coloboma and microphthalmia) at each morpholinodose (5 or 10 ng) and time-point studied (48, 72, 96 hpf). Forinstance, at a 5 ng gdf6a MO dose, 17% of 96 hpf morphantsexhibited skeletal anomalies compared to 82% with ocularanomalies (Fig. 3C) (n ¼ 96, x2, P , 0.001). Notably, adose-dependent effect was seen with both phenotypes andmorpholinos. To provide an additional control, a mismatch(mm) morpholino variant of gdf6aMO1 containing five nucleo-tide substitutions was injected at the same concentrations asgdf6aMO1 and gdf6aMO2, without yielding ocular or skeletalphenotypes (Fig. 3A and B).

In view of the markedly different prevalences of gdf6aMO-induced skeletal and ocular anomalies, and the incom-plete penetrance observed with A249E, Q253L and P327H,we next investigated the level of gdf6a mRNA in phenotypi-cally normal zebrafish morphants. Wild-type and morphantgdf6a zebrafish of 48 hpf were collected and divided intothree groups (wild-type, morphants exhibiting a phenotype,and phenotypically normal morphants) based on objectivemicroscopic appearance. Fifty embryos from each groupwere pooled, RNA extracted, cDNA prepared and gdf6amRNA variants amplified as described elsewhere (2).Observation of appreciable reductions in the level of correctlyspliced gdf6a mRNA in phenotypically unaffected morphantembryo pools (Fig. 3D) demonstrate that zebrafish withreduced levels of gdf6a mRNA may appear phenotypicallynormal.

Further characterization of morpholino-induced zebrafishskeletal phenotypes was undertaken to document the axialskeletal changes in gdf6a morphants and permit study ofgenes whose expression may be regulated by GDF6 mutation.At 48 hpf, morphants exhibited kinked (mild), bent (moderate)and curly (severe) tail phenotypes, compared with the straighttails of wild-type zebrafish (Fig. 3E–H). Whole-mount in situhybridization were undertaken on wild-type and gdf6a mor-phant zebrafish (at 10 somites, 18 hpf and 2 dpf) usingdigoxigenin-labeled antisense RNA probes to somite markersmyod, her7 and unc45; paralog gdf5; and BMP antagonists,

gremlin1, noggin1 and noggin2. No significant differences inthe expression of myod, her7 and unc45 were observed (datanot shown), however, expression of gdf5 was reduced in thedeveloping axial jaw and gill cartilage elements in gdf6a mor-phants (Fig. 3I and J) with gdf6a not required for gdf5expression in lateral jaw cartilage elements. Expression ofnoggin1 and noggin2 (57), were reduced in the newlyformed caudal somites of gdf6a morphants (92% [23/25])and the ventral aspects of the somites of gdf6a morphants(94% [17/18]), respectively (Fig. 3K–N). In situ observationswere validated by qPCR of noggin1 and noggin2; additionalcomparisons performed with noggin3 revealed significantlydecreased expression in gdf6a morphants compared with wild-type (Fig. 3O). Since expression of gremlin1 in the nasal retinais strongly reduced in gdf6a morphants (Fig. 3Q), both humanorthologs (GREMLIN1, GREMLIN2) and the related BMPantagonist, NOGGIN, were sequenced in 96 MAC patientswithout any mutations being identified (data not shown).

Murine model

In order to study a mammalian model organism, Gdf6þ/2 mice(Mouse Genome Informatics [MGI: 3604391]), were nextexamined. Analysis of the 43 offspring generated by sevenGdf6þ/2

� Gdf6þ/2 crosses revealed non-Mendelian ratios[Gdf62/2 (n ¼ 1), Gdf6þ/2 (n ¼ 25), Gdf6þ/þ (n ¼ 17)] andreduced litter size (mean, n ¼ 6). Variable and asymmetricocular Gdf6þ/2 phenotypes were observed, including: opticdisc excavation (eight of 12), microphthalmia (one of 12)and marked asymmetry (six of 12) (Fig. 4A–F). Ocular his-tology revealed scleral canal enlargement in Gdf6þ/2 mice(n ¼ 6) that corresponded with the clinically apparentin vivo optic nerve head cupping (Fig. 4G and H). Photopicand scotopic electroretinograms did not demonstrate any sig-nificant difference in the amplitude (a- and b-wave) or implicittimes (latency) between wild-type and Gdf6þ/2 mice (n ¼ 14)(data not shown). Two Gdf6þ/2 mice were screened for skel-etal phenotypes using high-resolution micro-CT, however, noappreciable differences were evident at 2 months of age com-pared with wild-type littermates and in particular, no vertebralfusions were present.

DISCUSSION

Although our data provide clear evidence for GDF6’s role inocular and skeletal development, certain features are incompati-ble with simple Mendelian inheritance. Incomplete penetranceand species-specific discrepancies in GDF6-attributable pheno-types were revealed by integrating analyses of a large patientcohort with two animal models. Such features, as well as pheno-typic differences at the level of individual mutations, and in onecase with the same mutation on different genetic backgrounds,provide evidence of more complex genetic mechanisms.

Incompletely penetrant phenotypes associated with threeseparate GDF6 mutations demonstrate that a single mutantallele can be insufficient to cause disease, a view supportedby analogous findings from zebrafish gdf6a morphants witha common genetic background (Fig. 3D). Considerablereliance can be placed on these findings because of the


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profound human ocular phenotypes that are incompatible withincomplete ascertainment, corroborative findings from lucifer-ase and Western analyses of GDF6 mutations, and dominantlyinherited Gdf6 murine phenotypes (35). This intra-familialvariability is highly relevant to human health since elucidatingthe underlying epistatic interactions would enhance under-standing of complex genetic mechanisms, improve geneticcounseling for affected pedigrees and, in the longer term,offer potential for modulating these effects therapeutically.However, the complexity of the BMP signaling pathwayrepresents one challenge to deciphering the mechanism(s)involved.

BMPs are synthesized as pro-proteins that are sequentiallycleaved and processed to yield disulfide-linked dimers. Afterbinding to type II and type I BMP receptors, the heterotetra-meric receptor complex results in phosphorylation and acti-vation of consecutive tiers of downstream SMADs (58,59).This multi-step, indirect and non-linear pathway, in whichindividual receptors subserve multiple ligands, providesscope for factors modulating ligand activity. The extensivefunctional redundancy of BMP signaling is well recognized(60) and includes large numbers of paralogs, antagonists andreceptors (25,61) as well as intracellular inhibitors (62,63).In this context, observation of altered gdf5 expression in

skeletal elements of gdf6a zebrafish morphants (Fig. 3I and J)accords with close paralogs possessing potentially relatedskeletal functions. Evidence that BMP antagonists are simi-larly affected by decreased gdf6 function is provided byaltered gremlin (ocular) (Fig. 3P and Q) and noggin 1 andnoggin 2 (skeletal) expression (Fig. 3K–N). The contributionof such genes to ocular phenotypes remains to be definedalthough screening of a cohort of 96 MAC DNA samplesfor mutations in these (three of 13) BMP antagonists did notidentify any significant sequence changes. Other genesrecently shown to modulate the BMP signaling pathwayinclude: co-receptors DRAGON and RGMa that facilitateligand-binding (64,65); murine convertase Pcsk5, whichcleaves the pro-domain from mature Gdf11 ligand (66); andhistone deacetylase 3 (67). Taken together with the key evolu-tionarily conserved role that gradients of BMP activity have inpatterning the vertebrate’s dorso-ventral axis, it is thus plaus-ible that buffering by parologs/BMP antagonists and othergenes may compensate for mildly perturbed GDF6 function,and combined with stochastic effects ensure that diseasephenotypes do not manifest.

In addition to incomplete penetrance, variable phenotypeswere observed in nine probands and in both zebrafish andmurine models. Patients exhibited either ocular, skeletal or

Figure 3. Zebrafish phenotypes induced by gdf6a MO inhibition. Summary of ocular (A) and skeletal (B) phenotypes in the gdf6a morphant zebrafish, illustratingthat the prevalence of these phenotypes differ markedly and is gdf6a MO dose-dependent (C). (D) RT-PCR of mRNA from pooled wildtype (WT) andgdf6aMO1-injected embryos illustrate similar reduction in the level of spliced gdf6 product in morphants with (þ) and without (2) skeletal and ocular phenotypes(control, elongation factor 1 alpha). Compared to wildtype (E), a range of mild (F), moderate (G) and severe (H) phenotypes were observed. Compared towildtype (I), in situ hybridization demonstrates reduced gdf5 expression in the developing jaw of gdf6a morphants (J). Reduced expression of noggin1 (Kand L) and noggin2 (M and N) were observed in the somites of gdf6a morphants (L, N) as compared to the wildtype (K, M). (O) RT-qPCR resultsshowing a reduction of the expression of antagonists noggin1, noggin2 and noggin3 in gdf6a morphants compared to wildtype. Expression of the BMP antagonistgremlin 1 in the zebrafish nasal retina is strongly reduced in gdf6a morphants (Q) when compared with wildtype (P).

Figure 4. Ocular phenotypes of Gdf6þ/2 mice. (A–E) Retinal in vivo photography illustrating the spectrum of optic nerve head changes present in Gdf6þ/2

mice. Note the severe optic disc excavation (B) when compared with wild-type (A) and variable fundus morphology in the Gdf6þ/2 mice (C–E). (F)Reduced ocular size in a Gdf6þ/2 mouse corresponds to human microphthalmia. Representative histological sections from wild-type (G) and Gdf6þ/2 mice(H) illustrating the enlarged scleral canal, consistent with clinically apparent optic cupping.


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oculo-skeletal anomalies (Table 1). The former represent partof a heterogeneous developmental spectrum, in which unilat-eral or bilateral disease, variation in ocular size (microphthal-mia) and embryonic fissure closure defects (coloboma) may bepresent (68,69), whereas the skeletal anomalies encompassspondylothoracic dysostosis, cervical and rib fusions, hemi-vertebrae and post-axial polydactyly. Although comparableocular and skeletal defects are present in gdf6a zebrafish mor-phants, the prevalence of each differed markedly (Fig. 3A–C).Even though no patient had both vertebral fusions and MAC(Table 1) (2), a subset of morphants exhibited both pheno-types. The Gdf6þ/2 mice studied exhibited variable and asym-metric ocular phenotypes (Fig. 4) with interesting parallels tothe optic nerve cupping seen in glaucoma patients. However,preliminary analysis indicated that no significant skeletalchanges were present, and in particular no vertebral fusionswere apparent on either micro-CT or MRI. Since theseresults contrast with those from a second Gdf6þ/2 strainwhere skeletal but no ocular anomalies were reported (35),the implication is that genetic background may influencewhich phenotypes predominate in each species (70–72).This is supported by the differing phenotypes caused byA249E mutations, a likely mutational hotspot owing to thehigh (85%) adjacent GC-content (73,74). These phenotypescomprise either microphthalmia, coloboma and post-axialpolydactyly, or KF; and as at least two of the three probandsare unrelated (Fig. 1H and I), different phenotypes can begenerated by the same mutation. Although we first reportedmutations in GDF6 in two patients with vertebral fusions[ARVO meeting, Fort Lauderdale, USA, 2007] (49), whilstthis manuscript was under revision, mutations were alsoreported in KF (50). This publication is helpful in demon-strating that the disease phenotype in the large pedigreefrom which proband 3 is derived only exhibits axial skeletaldisease, and that this segregates in an autosomal dominantmanner.

The seven mutations identified by screening a large patientcohort, demonstrate that approximately 1% and 4% of theocular and skeletal phenotypes studied are attributable toGDF6, in keeping with the genetic heterogeneity of these dis-orders (36,75–82). The expression pattern apparent on in situhybridization (data not shown) correlates with the phenotypicspectrum observed. Several strands of evidence demonstratethat the amino acid alterations observed represent pathogenicmutations, including the high degree of evolutionary conserva-tion evident in six of the seven mutations, their absence from alarge number of control chromosomes (Table 1) and the SNPdatabase, together with the complementary assays used tocharacterize a representative subset of mutations. The combi-nation of significantly reduced reporter activity compared withwild-type GDF6 (Fig. 2A) accords with the reduced levels ofmature ligand detected in the mutants by western blot analysis(Fig. 2B), indicating that A249E and K424R represent hypo-morphic mutations. Although biochemical characterization ofevery mutation is impracticable, the findings from A249E,affecting the least conserved residue (Fig. 1B) indicate thatalterations affecting more invariant residues are likely to havecomparable effects. The distribution of mutations throughoutthe pro- and mature domains correlates with findings inGDF5 (21,23,24,83–85) and contrasts with localization of

mutations in just the prodomain of BMP4 (17) and BMP15(18,19,86,87). The absence of frameshifts or truncations iscompatible with such mutations either resulting in phenotypesnot represented in the DNA collections screened, or alterna-tively, by dimerizing with the normal allele and inducingnonsense-mediated decay, resulting in dominant negativeeffects that are incompatible with viability [as seen withBMP15 (ovarian dysgenesis 2) (88) and BMPR1B (A2 brachy-dactyly) (15)]. Although our data do not allow us to differen-tiate between these possibilities, the non-Mendelian ratios ofGdf62/2 mice (1 of 43) imply the absence of GDF6 functionresults in reduced viability.

Other interesting features of our data include evidence thatGDF6 may underlie a broader range of phenotypes. Twopatients were found to have additional systemic anomalies:proband 4 (K424R) had a fused (horseshoe) kidney in additionto rib fusions and hemi-vertebrae, whereas proband 5 (Q253L)had a single testis, in addition to microphthalmia. Such find-ings accord with BMPs’ roles in renal (89,90) and gonadal(91) development and suggest that in addition to highly vari-able ocular or skeletal disorders, GDF6 may also underlie aspectrum of seemingly sporadic anomalies in other organs.Combined with altered gdf5 expression observed in gdf6amorphants in skeletal elements (Fig. 3G and H), this raisesthe possibility that GDF6 mutations may cause a broaderrange of disorders. Indeed, a locus for non-syndromiccleft lip and palate has recently been mapped to a regionencompassing GDF6 (90). In view of the increase inGDF5-attributable phenotypes that have been identified overthe last few years [brachydactyly type A1 (24), brachydactylytype C (22), acromesomelic dysplasia (20), chrondrodysplasia(21), symphalangism (24) and multiple synostosis syndrome 2(33)], the spectrum of phenotypes ascribed to GDF6 isexpected to expand. Finally, where family data were available,segregation of ocular and skeletal phenotypes appear dominantand fully or incompletely penetrant (Table 1). This accordswith the presence of ocular defects in haploinsufficient Gdf6mice (Fig. 4) and in a patient with a GDF6-encompassing del-etion (2), as well as reported dominant inheritance of A249Eand an 8q22.2-22.3 inversion in KF pedigrees (50). Interest-ingly, screening revealed no truncation mutations (missense,nonsense) or homozygous mutations.

In summary, these experiments identify seven GDF6mutations; four in patients with ocular anomalies and threeassociated with skeletal phenotypes. The pleiotropic effects(ocular and/or skeletal) of the specific mutations in combi-nation with variable expressivity (coloboma/microphthalmiaor vertebral fusion/hemi-vertebrae) and variable penetrance(full/incomplete) describe a novel and complex non-Mendelian inheritance pattern for the corresponding diseases.Our data demonstrate that GDF6 mutations account for 1% ofMAC and 4% of vertebral fusion cases implicating perturbedTGF-b signaling in a proportion of ocular and skeletal dis-orders, which helps identify candidates from the largeTGF-b family that merit further investigation. Through thealtered noggin1, noggin2, noggin 3, gremlin1 and gdf5expression observed in gdf6a morphants, we begin to definegenes downstream of GDF6/gdf6a. By extendingGDF6-attributable phenotypes to other organ systems, thesefindings provide a potential explanation for some of the


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reported pedigrees with inherited oculo-skeletal disease. Inlight of gdf6a’s role as the key determinant of zebrafish dorso-ventral patterning (92), we hypothesize that this gene pos-sesses a related function in higher vertebrates and thus maybe responsible for a wider range of human diseases beyondskeletal and ocular disorders. An approach integrating ana-lyses of patients and animal models of impaired GDF6 func-tion may aid in elucidating this gene’s broader functions aswell as the factors mediating penetrance and phenotypic var-iance that are significant to the pathogenesis of human disease.

MATERIALS AND METHODS

Patients

DNA was obtained from blood samples of patients with ocularanomalies (n ¼ 489) and vertebral segmentation anomalies(n ¼ 81) from six centers in four countries. Four patients fromthe screening panel exhibited both ocular (Duane’s syndrome,strabismus, coloboma and visual impairment) and skeletal(polydactyly, vertebral fusions, hemi-vertebrae) phenotypes.Vertebral defects were determined by physical examination,supplemented by radiography in affected individuals. Ethicalapproval for this study was obtained from the University ofAlberta Hospital Health Research Ethics Board, and informedconsent was obtained from all participants.

Mutation analysis

Three pairs of primers amplifying the two exons of GDF6were designed using Primer3 (http://www.broad.mit.edu/cgi-bin/primer/primer3_www.cgi) and sequences availablefrom Ensembl (http://www.ensembl.org/index.html) andNCBI (http://www.ncbi.nih.gov) (primer sequences and con-ditions available on request). Briefly, genomic DNA (50 ng)was amplified with Taq polymerase with 10% glycerol and5% formamide at an annealing temperature of 578C usingstandard methods. Amplicons spanning both exons weresequenced on an ABI Prism 3100 capillary sequencer(Applied Biosystems, Foster City, CA, USA) and the dataanalyzed using Sequencher 4.5 software (GeneCodes,Madison, WI, USA). BsrBI and Tsp509I restriction enzymeswere used to screen 646 control chromosomes for A249Eand K424R mutations, respectively, whereas 366 controlchromosomes were screened with the following enzymes:HpyCH4V (Q253L and P327H), SphI, (G42V), BsrI (Q119R)and TaqaI (D216G). Digestion products were scored followingelectrophoresis on a 1% agarose gel with ethidium bromide.

Quantitative polymerase chain reaction and array CGH

qPCR was used to screen a subset of 32 patients for copynumber alterations of GDF6 (Taqman, Applied Biosystems).Primers were designed using Primer Express software (ABI)(available on request) and qPCR was performed togetherwith connexin 40 (TaqMan Gene Expression Assay ID:Hs99999170_s1; Applied Biosystems) as an internal control.Samples were cycled 40 times at 958C for 15 s and 608C for1 min (ABI Prism, 7000, Sequence Detection System). ADNA sample from the proband of the SHFM pedigree that

maps to 8q21.11-q22.3 was hybridized to an oligonucleotidearray comprising 385,000 probes at a mean spacing of 6-kbas described elsewhere (46).

Reporter gene assay

After adding a Kozak consensus sequence to permit ribosomalbinding on wild-type, A249E and K424R human GDF6cDNA, sequences were initially cloned into TOPO4 (Invitro-gen), then CS2 vector and 1–200 pg of capped, poly-A tailedmRNA (mMessage mMachinew, Ambion Inc, Austin, TX,USA) were injected into 1-cell embryos. For the biochemicalassay, PLM cells were harvested from embryonic age (E) 11.5CD-1 mouse embryos as previously described (51,52). Transfec-tions were carried out with Effectene (Qiagen, Mississauga, ON,CAN) according to the manufacturer’s instructions in 384-wellplates. Briefly, DNA-transfection mixtures were aliquoted intowells followed by the addition of approximately 100,000 PLMcells, and wells were topped-up to a total volume of 100 ml.Media was replenished 24 h post-transfection, and lysateswere collected 48 h post-transfection. Luciferase activity wasmeasured using the Dual Luciferase Kit (Promega, Madison,WI, USA) and firefly luciferase was normalized to an internalRenilla luciferase control. Luciferase assays were performed inquadruplicate and repeated three times.

Western blot analysis

The coding sequence of GDF6 and mutations A249E andK424R (minus the stop codon) were incorporated intoGateway ENTR-D-TOPO, integrated into mammalianV5-tagged Destination vector (Invitrogen, Carlsbad, CA,USA), and confirmed by sequencing. Transient transfections inCOS7 cells were performed with FuGENE (Roche, Diagnostics,Indianapolis, IN, USA) according to manufacturer’s instructionson 100 mm plates with culture media and lysates collected 48 hpost-transfection as previously described (93). Proteins wereextracted, separated on a 15% SDS–PAGE gel and transferredto nitrocellulose membranes (BioRad, Hercules, CA, USA),which were incubated with anti-V5 (1:10,000), secreted alkalinephosphatase (1:5000) or a-tubulin (1:10,000) primary antibody(AbCam, Cambridge, MA, USA), and subsequently with anti-mouse or anti-rabbit IgG-HRP (1:5000, Jackson Laboratories,West Grove, PA, USA). The antibodies were detected bychemiluminescence (Pierce, ThermoScientific, Rockford, IL,USA). b-Galactosidase was co-transfected with GDF6þV5vectors and examined with a b-galactosidase enzyme assay(Promega) for transfection control.

Zebrafish phenotyping and in situ hybridization

Zebrafish (AB strain) knockdown experiments were performedas described earlier (2), with an additional gdf6a morpholinocontaining five nucleotide substitutions as a control (sequencesavailable on request). RNA whole-mount in situ hybridizationsfor myod, her7, unc45, nog1, nog2, gremlin1 and gdf5 wereperformed as previously described (94). Stained embryoswere mounted in 70% glycerol and photographed with aQimaging micropublisher digital charge-coupled devicecamera. For qPCR validation of in situ hybridizations,


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zebrafish embryos were collected at 24 h post-fertilization,total RNA extracted (RNaqueous, Ambion, Foster City, CA,USA) and cDNA synthesized (Stratagene, La Jolla, CA,USA). Primers were designed for noggin1, noggin2 andnoggin3 using the ‘Roche Applied Science Universal ProbeLibrary’ https://www.roche-applied-science.com/sis/rtper/upl/index.jsp (available on request). Expression levels werequantified by qPCR using SYBR Green chemistry (Stratagene),where ef1a was used as a control. All PCRs were performedtwice in triplicate.

Murine genotyping and phenotyping

Gdf6þ/2 mice [strain Gdf6tm1Lex (MGI:3604391) http://www.informatics.jax.org] were housed and handled in accordancewith the University of Alberta Animal Policy and WelfareCommittee protocols. Mice were genotyped using allele-specific primers (sequences and conditions available onrequest) on DNA derived from ear-notched tissue. Micewere anaesthetized using isoflurane and pupils were dilatedwith Tropicamide (Alcon, USA). Images were initially capturedwith a digital camera through an OPMIw VISU 160 microscope(Zeiss, Germany). Subsequent examinations were undertakenusing an endoscope with an attached otoscope (1218AA;Karl Storz, Tuttlingen, Germany) (95) and still images andvideos were captured on a Telecam SLII camera (reference202130-20, Karl Storz) with a Xenon lamp light source(481C, Karl Storz) and processed with Pinnacle StudioTM

software (Avid Technology, Inc., MA, USA).Micro-CT imaging was performed on a Microtomograph

1076 (Skyscan NV, Aartselaar, Belgium). Serial cross-sectional images were produced of isotropic 18 mm3 voxels,from the 1808 angular rotational scans (0.58 incrementalsteps) and reconstructed using a modified Feldkamp algorithm(40). All image data were Gaussian-filtered and globallythresholded using standardized minimum and maximumcross-section to image conversion values of 0.0–0.0600,respectively, to extract the mineralized phase representingthe three-dimensional (3D) bone architecture. Qualitativeassessment of skeletal formation was performed from renderedvisualizations of the 3D architecture, while quantitative analysisof the vertebral bodies was undertaken with morphometricalanalysis software (CTan, Skyscan NV, Aartselaar, Belgium)for the bone volume ratio [volume of bone/total volume ofbone and soft tissue], as described elsewhere (41).

ACKNOWLEDGEMENTS

The authors thank the patients for participating in this study;Dr Fred Berry for very helpful discussions; Drs KarenTemple, David Fitzpatrick and Raymond Clarke for provisionof DNA samples; and Anthony Lott, Helen Chung, MathewLaroque, May Yu, Tim Footz, Yoko Ito, Hermina Strungau,Timothy Erickson, B. Hemadevi and B. Suganthalakshmi,Drs Stacey Bleoo, Martin Somerville and Gino Fallone fortheir assistance.

Conflicts of Interest statement. None declared.

FUNDING

The Canadian Institutes of Health Research (to O.J.L. andT.M.U.), Alberta Heritage Foundation for Medical Researchand Canadian Foundation for Innovation (to O.J.L.), NationalScientific Engineering Council (to C.R.F. and A.J.W.) andAlberta Ingenuity Fund (to A.J.W.). A.J.W. and O.J.L. arerecipients of Canada Research Chairs.

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Incomplete penetrance and phenotypic variability characterize Gdf6-attributable oculo-skeletal