Incomplete penetrance and phenotypic variability
characterize Gdf6-attributable oculo-skeletal
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 Sauve ´1,
Marc Abitbol4, Periasamy Sundaresan12, Veronica van Heyningen13, Olivier Pourquie ´5,
T. Michael Underhill3, Andrew J. Waskiewicz2and 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 ministe `re 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 and13MRC 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 skeletal
development; however, because of their widespread expression and functional redundancy, less progress
has been made identifying the roles of individual BMPs in human disease. We identified seven heterozygous
mutations in growth differentiation factor 6 (GDF6), a member of the BMP family, in patients with both ocular
and vertebral anomalies, characterized their effects with a SOX9-reporter assay and western analysis, and
demonstrated 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 formation
and altered expression of somite markers noggin1 and noggin2. Gdf61/2mice exhibited variable ocular
phenotypes compatible with phenotypes observed in patients and zebrafish. Key differences evident
between patients and animal models included pleiotropic effects, variable expressivity and incomplete
penetrance. 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 BMP
function and its contributions to human disease.
?To whom correspondence should be addressed. Tel: þ1 780 492 8550; Fax: þ1 780 492 6934; Email: firstname.lastname@example.org
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Human Molecular Genetics, 2009, Vol. 18, No. 6
Advance Access published on January 6, 2009
The Growth Differentiation Factors (GDFs) are members
of the bone morphogenetic proteins (BMP) sub-family of
transforming growth factor-beta (TGF-b) signaling ligands,
known to regulate patterning during development (1). We
previously demonstrated GDF6’s role in ocular development
by characterizing a GDF6-encompassing copy number
variant in a patient with ocular colobomata, and recapitulating
the human phenotype in gdf6a morphant zebrafish (2). Such
results, combined with comparable findings in Xenopus (3),
suggested that GDF6 mutations may underlie a range
of ocular phenotypes, including one or more components of
the microphthalmia, anophthalmia or coloboma (MAC)
developmental spectrum. In addition, in keeping with
the broad and incompletely defined roles of BMPs, it was
probable that GDF6 mutations contributed to other human
The BMPs were originally identified through their capacity
to induce bone formation (4,5) and are now recognized to have
critical roles patterning a diverse range of tissues including
bone, heart, lungs and kidney (6–10). Over the last 10 years
significant progress characterizing the phenotypes resulting
from mutation of BMPs or their receptors has ascribed
human 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 type
II BMP receptors [ACVR1 (11), ACVRL1 (12), BMPR1A
(13), TGFBR1 (14), BMPR1B (15), BMPRII (16)] rather
than signaling ligands [BMP4 (17), BMP15 (18,19), GDF5
(20–24)]. This distribution likely reflects the challenges dis-
cerning human disease phenotypes for ligands that exhibit
functional redundancy (25). The contrast between the limited
human phenotypes of individual ligands compared with the
broad expression pattern and expansive range of phenotypes
associated with BMP loss in model organisms implies that
only a fraction of the human phenotypes attributable to each
have been identified. This is clearly illustrated by the data
from BMP4, where human mutations are primarily associated
with microphthalmia (17), while murine loss of function
causes defects in multiple systems: cardiovascular (26,27),
craniofacial (28), ocular (25,29), reproductive, limbs, digits
(30) and auditory (31). Increasing the proportion of BMPs
with a defined function is thus of dual clinical and scientific
importance—advancing understanding of the molecular basis
of human disease, and in view of the disparate early and
late-onset functions of individual BMPs (20,23,24,32–34),
offering potential to inform understanding of multiple
aspects of human genetics.
Accordingly, we set out to define GDF6’s function in
greater detail and present data demonstrating a broad role in
human disease. At the outset of our studies, two lines of evi-
dence indicated that, like other BMPs, GDF6 at a minimum
possessed an appreciable skeletal developmental role. The
first was data from a murine model demonstrating Gdf6’s
regulation 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 and
foot malformation (SHFM) (37)] to the vicinity of GDF6
(Fig. 1A), was also compatible with a skeletal function. KF
syndrome, characterized by defective cervical, thoracic or
lumbar vertebral segmentation (38), is frequently associated
with other skeletal (scoliosis, rib abnormalities, Sprengel’s
deformity) and non-skeletal (deafness, cardiovascular, ocular
and renal) anomalies (39–41). Some of these (e.g. deafness
and rib anomalies) correlate with the extensive expression
pattern of Gdf6 (35). Furthermore, existence of a KF
subtype with an ocular phenotype [Wildervanck syndrome
(42)] is compatible with the possibility that GDF6 causes
both ocular and skeletal disease. The mapping of the sixth
SHFM (43) locus to a large GDF6-encompassing interval
(37) raised the possibility that this gene might underlie this
In light of these factors supporting GDF6 involvement in
human ocular and skeletal disorders, we screened patients
with ocular and skeletal developmental anomalies for GDF6
mutations. We investigated the biological significance of a
representative subset of the mutations identified with a repor-
ter gene assay and western blot analysis, and characterized the
effects of perturbed Gdf6 function in two model organisms
(murine and zebrafish). Our results indicate that GDF6
mutations result in variable ocular and skeletal phenotypes
with evidence of non-Mendelian inheritance, according
closely with comparable features in animal models of
decreased Gdf6 function. Such findings have implications
for deciphering interactions between members of the BMP
family and provide a model for studying the contribution of
complex inheritance patterns to human disease (44,45).
Sequencing and mutation analysis
DNA samples from 489 patients with ocular anomalies
(micropththalmia, anophthalmia and coloboma), and 81
patients with vertebral segmentation anomalies were screened
for GDF6 mutations by sequencing amplicons encompassing
the two exons and splice sites. A subset of 32 samples were
screened for copy number alteration by real-time quantitative
PCR (qPCR) (TaqManw), yielding normal results. Hybridiz-
8q21.11-q22.3 linked SHFM pedigree to a CGH array with a
mean 6 kb oligonucleotide probe spacing (46), identified no
copy number variations in the 3 Mb region encompassing
Sequencing identified two heterozygous sequence changes
in exon 1 (125 g!t; 356a!g) and five heterozygous
changes (647 g!a,
1271a!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 with
ocular phenotypes, two with skeletal phenotypes (G42V and
K424R), while one alteration (A249E) identified in three pro-
bands, was associated with either ocular or skeletal phenotypes
from theprobandof the
746c!a,758a!t, 980c!a and
Human Molecular Genetics, 2009, Vol. 18, No. 6 1111
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 and
GDF7. Photograph of unilateral microphthalmia (C and D) in an individual with heterozygous GDF6 mutation P327H, and corresponding fundus photographs (E
and F). (G) In three pedigrees a phenotypically unaffected parent (M1, F2 and M3) carries the mutation as illustrated by enzyme digestion (A249E, P327H, and
Q253L) and gel separation [P (proband), F (father), M (mother), C (control), L (Ladder)]. (H) Subset of chromosome 8q22 microsatellite markers used for
haplotype analysis of three patients carrying A249E and (I) the four informative microsatellite markers demonstrating Klippel-Feil proband 3, is unrelated to
coloboma proband 1 and microphthalmia proband 2.
Table 1. Summary of mutations identified in GDF6
Penetrance Enzyme (no. of controls)
‘–’ Denotes unknown because of the nature of DNA collection from which samples were derived.
1112 Human Molecular Genetics, 2009, Vol. 18, No. 6
The seven residues affected vary in their degree of conser-
vation, with two conserved in all vertebrates (K424R and
D216G), 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 the
ability to recall family members. However, for three of the
seven amino acid alterations additional DNA samples could
be obtained, allowing segregation to be assessed. In each of
these cases, A249E (proband 1), Q253L (proband 5) and
P327H (proband 6), the presence of the alteration in an unaf-
fected parent indicates incomplete penetrance (Fig. 1G).
Sequence and structure analyses demonstrate that K424R
affects 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 soluble
inhibitors. Activin A, another member of the BMP superfamily,
interacts with the minimal inhibitory module of follistatin
through multiple contacts mapped to this surface, with
charged residues in both molecules forming an extensive
network of hydrogen bonds (47). Replacement of this lysine
with a larger arginine residue increases the similarity of
GDF6 to GDF5 and GDF7 (members of the same clade as
GDF6) that have an arginine in this position, and may poten-
tially decrease the specificity of the interaction with GDF6’s
receptor(s). Similarly, the residue altered by D216G is invariant
in 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 identified
in this study, the A249E alteration affects the least conserved
residue and is located in the TGF-b prodomain, a region
thought to facilitate correct folding of the mature secreted
peptide (48). A249E was identified in three probands from
geographically disparate countries [Table 1; (49,50)] and
genotyping with eight microsatellites and six single nucleotide
polymorphism (SNPs) of which four were informative
(Fig. 1H), demonstrated that at least two of these individuals
carry different haplotypes (Fig. 1I).
Reporter gene and protein expression assays
To characterize the functional consequences of the amino acid
changes, two alterations (A249E and K424R) were selected
that lie in different GDF6 domains (Fig. 1B). A functional
assay employing a SOX9-responsive reporter was used to
evaluate BMP signaling. SOX9 plays a central role in chondro-
genesis, and as its expression/activity is exquisitely sensitive
to changes in BMP/GDF activity, this reporter gene provides
a reliable read-out on the status of BMP/GDF signaling
(51,52). Accordingly, to assess chondrogenic potential,
expression constructs for GDF6 and mutants A249E and
K424R were co-transfected with a SOX9-responsive reporter
gene into primary limb mesenchymal (PLM) cells and the
activities 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 increased
SOX9 reporter activity. In contrast, expression of GDF6–
A249E and GDF6–K424R constructs resulted in 2.9-fold
and 2.4-fold increases in reporter gene activity (P , 0.034
and 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 media
from wild-type and mutant GDF6 was next undertaken to
determine the effects of the two representative mutations on
protein expression and secretion. GDF6 cDNA was mutagen-
ized for the two mutations (A249E and K424R), tagged with a
V5 epitope, and transiently transfected into COS7 cells. Pro-
teins collected from the media and whole cell lysates were
separated by sodium dodecyl sulfate–polyacrylamide gel elec-
trophoresis, transferred to membranes and probed with V5
antibody. The western blot revealed the presence of
pro-GDF6 (55 kDa) in both media and cell lysates, with the
mature GDF6 ligand (approximately 16 kDa) present only in
the media as a doublet (Fig. 2B). Although neither mutant
constructs exhibited abnormal formation of pro-GDF6 nor
mature GDF6 protein, the level of secreted mature GDF6
was reduced with A249E (23%) and K424R (83%) mutants
compared with wild-type [ImageJ (53)] (Fig. 2B). Inclusion of
a-tubulin (whole cell lysate) and secreted alkaline phosphatase
Figure 2. Functional analyses of GDF6 mutants. (A) Significant differences in
activity of A249E and K424R-containing mutants in the SOX9-reporter luci-
ferase assay were observed, compared with wild-type. [Reporter gene activity
expressed in relative light units, normalized to the control; the percentage
reduction compared with wild-type are: A249E ¼ 14% (0.1098), K424R ¼
30% (0.0897); wild-type ¼ 0.1273]. (B) Western blot analysis of wild-type
GDF6 and mutants A249E and K424R. Both pro-GDF6 (55 kDa) and
mature GDF6 (15 kDa) were present in the supernatant (lanes 1–3), with
pro-GDF6 detected in the cell lysates (lanes 4–6). Reduced amounts of
mature GDF6 were observed in mutant GDF6 (lanes 2 and 3) when compared
with wild-type (lane 1). Secreted alkaline phosphatase and a-tubulin controls
demonstrate proper translation and secretion of control protein in supernatant
and cell lysates, respectively. (UN ¼ untransfected control).
Human Molecular Genetics, 2009, Vol. 18, No. 61113
(supernatants) provided controls for consistent loading and
secretion (Fig. 2B). Co-transfection of the GDF6–V5 con-
structs with b-galactosidase enzyme construct and subsequent
assay for b-galactosidase activity (Promega) revealed equal
V5-tagged GDF6 (data not shown).
In light of the variable human phenotypes observed with
GDF6 mutations (Fig. 1C–F, Table 1) and the incomplete
penetrance evident with three of seven mutations (Fig. 1G),
we studied a gdf6a zebrafish model to determine if comparable
features were present. gdf6a function was inhibited using mor-
pholino antisense oligonucleotides (MO). The first (gdf6aMO1)
targets the 50-splice site, while the second (gdf6aMO2) targets
the intron 1–exon 2 boundary (2). One cell zebrafish
embryos were injected with gdf6aMO1or gdf6aMO2at 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 reduce
apoptotic cell death (56). The prevalence of skeletal anomalies
(curled or kinked tails) was much lower than that of ocular
anomalies (coloboma and microphthalmia) at each morpholino
dose (5 or 10 ng) and time-point studied (48, 72, 96 hpf). For
instance, at a 5 ng gdf6a MO dose, 17% of 96 hpf morphants
exhibited skeletal anomalies compared to 82% with ocular
anomalies (Fig. 3C) (n ¼ 96, x2, P , 0.001). Notably, a
dose-dependent effect was seen with both phenotypes and
morpholinos. To provide an additional control, a mismatch
(mm) morpholino variant of gdf6aMO1containing five nucleo-
tide substitutions was injected at the same concentrations as
gdf6aMO1and gdf6aMO2, without yielding ocular or skeletal
phenotypes (Fig. 3A and B).
In view of the markedly different prevalences of gdf6a
MO-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 morphant
gdf6a zebrafish of 48 hpf were collected and divided into
three groups (wild-type, morphants exhibiting a phenotype,
and phenotypically normal morphants) based on objective
microscopic appearance. Fifty embryos from each group
were pooled, RNA extracted, cDNA prepared and gdf6a
mRNA variants amplified as described elsewhere (2).
Observation of appreciable reductions in the level of correctly
spliced gdf6a mRNA in phenotypically unaffected morphant
embryo pools (Fig. 3D) demonstrate that zebrafish with
reduced levels of gdf6a mRNA may appear phenotypically
Further characterization of morpholino-induced zebrafish
skeletal phenotypes was undertaken to document the axial
skeletal changes in gdf6a morphants and permit study of
genes 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 straight
tails of wild-type zebrafish (Fig. 3E–H). Whole-mount in situ
hybridization were undertaken on wild-type and gdf6a mor-
phant zebrafish (at 10 somites, 18 hpf and 2 dpf) using
digoxigenin-labeled antisense RNA probes to somite markers
myod, her7 and unc45; paralog gdf5; and BMP antagonists,
gremlin1, noggin1 and noggin2. No significant differences in
the expression of myod, her7 and unc45 were observed (data
not shown), however, expression of gdf5 was reduced in the
developing axial jaw and gill cartilage elements in gdf6a mor-
phants (Fig. 3I and J) with gdf6a not required for gdf5
expression in lateral jaw cartilage elements. Expression of
noggin1 and noggin2 (57), were reduced in the newly
formed 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 observations
were validated by qPCR of noggin1 and noggin2; additional
comparisons performed with noggin3 revealed significantly
decreased expression in gdf6a morphants compared with wild-
type (Fig. 3O). Since expression of gremlin1 in the nasal retina
is strongly reduced in gdf6a morphants (Fig. 3Q), both human
orthologs (GREMLIN1, GREMLIN2) and the related BMP
antagonist, NOGGIN, were sequenced in 96 MAC patients
without any mutations being identified (data not shown).
In order to study a mammalian model organism, Gdf6þ/2mice
(Mouse Genome Informatics [MGI: 3604391]), were next
examined. Analysis of the 43 offspring generated by seven
Gdf6þ/2? Gdf6þ/2crosses revealed non-Mendelian ratios
[Gdf62/2(n ¼ 1), Gdf6þ/2(n ¼ 25), Gdf6þ/þ(n ¼ 17)] and
reduced litter size (mean, n ¼ 6). Variable and asymmetric
ocular Gdf6þ/2phenotypes were observed, including: optic
disc 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/2mice
(n ¼ 6) that corresponded with the clinically apparent
in vivo optic nerve head cupping (Fig. 4G and H). Photopic
and scotopic electroretinograms did not demonstrate any sig-
nificant difference in the amplitude (a- and b-wave) or implicit
times (latency) between wild-type and Gdf6þ/2mice (n ¼ 14)
(data not shown). Two Gdf6þ/2mice were screened for skel-
etal phenotypes using high-resolution micro-CT, however, no
appreciable differences were evident at 2 months of age com-
pared with wild-type littermates and in particular, no vertebral
fusions were present.
Although our data provide clear evidence for GDF6’s role in
ocular and skeletal development, certain features are incompati-
ble with simple Mendelian inheritance. Incomplete penetrance
and species-specific discrepancies in GDF6-attributable pheno-
types were revealed by integrating analyses of a large patient
cohort with two animal models. Such features, as well as pheno-
typic differences at the level of individual mutations, and in one
case with the same mutation on different genetic backgrounds,
provide evidence of more complex genetic mechanisms.
Incompletely penetrant phenotypes associated with three
separate GDF6 mutations demonstrate that a single mutant
allele can be insufficient to cause disease, a view supported
by analogous findings from zebrafish gdf6a morphants with
a common genetic background (Fig. 3D). Considerable
reliance can be placed on these findings because of the
1114Human Molecular Genetics, 2009, Vol. 18, No. 6
Human Molecular Genetics, 2009, Vol. 18, No. 61115
profound human ocular phenotypes that are incompatible with
incomplete ascertainment, corroborative findings from lucifer-
ase and Western analyses of GDF6 mutations, and dominantly
inherited Gdf6 murine phenotypes (35). This intra-familial
variability is highly relevant to human health since elucidating
the underlying epistatic interactions would enhance under-
standing of complex genetic mechanisms, improve genetic
counseling for affected pedigrees and, in the longer term,
offer potential for modulating these effects therapeutically.
However, the complexity of the BMP signaling pathway
represents one challenge to deciphering the mechanism(s)
BMPs are synthesized as pro-proteins that are sequentially
cleaved and processed to yield disulfide-linked dimers. After
binding 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 which
individual receptors subserve multiple ligands, provides
scope for factors modulating ligand activity. The extensive
functional redundancy of BMP signaling is well recognized
(60) and includes large numbers of paralogs, antagonists and
receptors (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 related
skeletal functions. Evidence that BMP antagonists are simi-
larly affected by decreased gdf6 function is provided by
altered gremlin (ocular) (Fig. 3P and Q) and noggin 1 and
noggin 2 (skeletal) expression (Fig. 3K–N). The contribution
of such genes to ocular phenotypes remains to be defined
although screening of a cohort of 96 MAC DNA samples
for mutations in these (three of 13) BMP antagonists did not
identify any significant sequence changes. Other genes
recently shown to modulate the BMP signaling pathway
include: co-receptors DRAGON and RGMa that facilitate
ligand-binding (64,65); murine convertase Pcsk5, which
cleaves the pro-domain from mature Gdf11 ligand (66); and
histone deacetylase 3 (67). Taken together with the key evolu-
tionarily conserved role that gradients of BMP activity have in
patterning the vertebrate’s dorso-ventral axis, it is thus plaus-
ible that buffering by parologs/BMP antagonists and other
genes may compensate for mildly perturbed GDF6 function,
and combined with stochastic effects ensure that disease
phenotypes do not manifest.
In addition to incomplete penetrance, variable phenotypes
were observed in nine probands and in both zebrafish and
murine 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, illustrating
that the prevalence of these phenotypes differ markedly and is gdf6a MO dose-dependent (C). (D) RT-PCR of mRNA from pooled wildtype (WT) and
gdf6aMO1-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 to
wildtype (I), in situ hybridization demonstrates reduced gdf5 expression in the developing jaw of gdf6a morphants (J). Reduced expression of noggin1 (K
and 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 results
showing a reduction of the expression of antagonists noggin1, noggin2 and noggin3 in gdf6a morphants compared to wildtype. Expression of the BMP antagonist
gremlin 1 in the zebrafish nasal retina is strongly reduced in gdf6a morphants (Q) when compared with wildtype (P).
Figure 4. Ocular phenotypes of Gdf6þ/2mice. (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/2mice (C–E). (F)
Reduced ocular size in a Gdf6þ/2mouse corresponds to human microphthalmia. Representative histological sections from wild-type (G) and Gdf6þ/2mice
(H) illustrating the enlarged scleral canal, consistent with clinically apparent optic cupping.
1116Human Molecular Genetics, 2009, Vol. 18, No. 6
oculo-skeletal anomalies (Table 1). The former represent part
of a heterogeneous developmental spectrum, in which unilat-
eral or bilateral disease, variation in ocular size (microphthal-
mia) and embryonic fissure closure defects (coloboma) may be
present (68,69), whereas the skeletal anomalies encompass
spondylothoracic dysostosis, cervical and rib fusions, hemi-
vertebrae and post-axial polydactyly. Although comparable
ocular 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/2mice studied exhibited variable and asym-
metric ocular phenotypes (Fig. 4) with interesting parallels to
the optic nerve cupping seen in glaucoma patients. However,
preliminary analysis indicated that no significant skeletal
changes were present, and in particular no vertebral fusions
were apparent on either micro-CT or MRI. Since these
results contrast with those from a second Gdf6þ/2strain
where skeletal but no ocular anomalies were reported (35),
the implication is that genetic background may influence
which phenotypes predominate in each species (70–72).
This is supported by the differing phenotypes caused by
A249E mutations, a likely mutational hotspot owing to the
high (85%) adjacent GC-content (73,74). These phenotypes
comprise either microphthalmia, coloboma and post-axial
polydactyly, or KF; and as at least two of the three probands
are unrelated (Fig. 1H and I), different phenotypes can be
generated by the same mutation. Although we first reported
mutations in GDF6 in two patients with vertebral fusions
[ARVO meeting, Fort Lauderdale, USA, 2007] (49), whilst
this manuscript was under revision, mutations were also
reported in KF (50). This publication is helpful in demon-
strating that the disease phenotype in the large pedigree
from which proband 3 is derived only exhibits axial skeletal
disease, and that this segregates in an autosomal dominant
The seven mutations identified by screening a large patient
cohort, demonstrate that approximately 1% and 4% of the
ocular and skeletal phenotypes studied are attributable to
GDF6, in keeping with the genetic heterogeneity of these dis-
orders (36,75–82). The expression pattern apparent on in situ
hybridization (data not shown) correlates with the phenotypic
spectrum observed. Several strands of evidence demonstrate
that the amino acid alterations observed represent pathogenic
mutations, including the high degree of evolutionary conserva-
tion evident in six of the seven mutations, their absence from a
large number of control chromosomes (Table 1) and the SNP
database, together with the complementary assays used to
characterize a representative subset of mutations. The combi-
nation of significantly reduced reporter activity compared with
wild-type GDF6 (Fig. 2A) accords with the reduced levels of
mature ligand detected in the mutants by western blot analysis
(Fig. 2B), indicating that A249E and K424R represent hypo-
morphic mutations. Although biochemical characterization of
every mutation is impracticable, the findings from A249E,
affecting the least conserved residue (Fig. 1B) indicate that
alterations affecting more invariant residues are likely to have
comparable effects. The distribution of mutations throughout
the pro- and mature domains correlates with findings in
GDF5 (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 is
compatible with such mutations either resulting in phenotypes
not represented in the DNA collections screened, or alterna-
tively, by dimerizing with the normal allele and inducing
nonsense-mediated decay, resulting in dominant negative
effects that are incompatible with viability [as seen with
BMP15 (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 of
Gdf62/2mice (1 of 43) imply the absence of GDF6 function
results in reduced viability.
Other interesting features of our data include evidence that
GDF6 may underlie a broader range of phenotypes. Two
patients were found to have additional systemic anomalies:
proband 4 (K424R) had a fused (horseshoe) kidney in addition
to 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 a
spectrum of seemingly sporadic anomalies in other organs.
Combined with altered gdf5 expression observed in gdf6a
morphants in skeletal elements (Fig. 3G and H), this raises
the possibility that GDF6 mutations may cause a broader
range of disorders. Indeed, a locus for non-syndromic
cleft lip and palate has recently been mapped to a region
encompassing GDF6 (90). In view of the increase in
GDF5-attributable phenotypes that have been identified over
the last few years [brachydactyly type A1 (24), brachydactyly
type C (22), acromesomelic dysplasia (20), chrondrodysplasia
(21), symphalangism (24) and multiple synostosis syndrome 2
(33)], the spectrum of phenotypes ascribed to GDF6 is
expected to expand. Finally, where family data were available,
segregation of ocular and skeletal phenotypes appear dominant
and fully or incompletely penetrant (Table 1). This accords
with the presence of ocular defects in haploinsufficient Gdf6
mice (Fig. 4) and in a patient with a GDF6-encompassing del-
etion (2), as well as reported dominant inheritance of A249E
and 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 GDF6
mutations; four in patients with ocular anomalies and three
associated with skeletal phenotypes. The pleiotropic effects
(ocular and/or skeletal) of the specific mutations in combi-
nation with variable expressivity (coloboma/microphthalmia
or vertebral fusion/hemi-vertebrae) and variable penetrance
Mendelian inheritance pattern for the corresponding diseases.
Our data demonstrate that GDF6 mutations account for 1% of
MAC and 4% of vertebral fusion cases implicating perturbed
TGF-b signaling in a proportion of ocular and skeletal dis-
orders, which helps identify candidates from the large
TGF-b family that merit further investigation. Through the
altered noggin1, noggin2, noggin 3, gremlin1 and gdf5
expression observed in gdf6a morphants, we begin to define
genes downstream of
GDF6-attributable phenotypes to other organ systems, these
findings provide a potential explanation for some of the
novel andcomplex non-
Human Molecular Genetics, 2009, Vol. 18, No. 6 1117
reported pedigrees with inherited oculo-skeletal disease. In
light 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 may
be responsible for a wider range of human diseases beyond
skeletal 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 as
well as the factors mediating penetrance and phenotypic var-
iance that are significant to the pathogenesis of human disease.
MATERIALS AND METHODS
DNA was obtained from blood samples of patients with ocular
anomalies (n ¼ 489) and vertebral segmentation anomalies
(n ¼ 81) from six centers in four countries. Four patients from
the 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. Ethical
approval for this study was obtained from the University of
Alberta Hospital Health Research Ethics Board, and informed
consent was obtained from all participants.
Three pairs of primers amplifying the two exons of GDF6
were designed using Primer3 (http://www.broad.mit.edu/
cgi-bin/primer/primer3_www.cgi) and sequences available
from Ensembl (http://www.ensembl.org/index.html)
NCBI (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 and
5% formamide at an annealing temperature of 578C using
standard methods. Amplicons spanning both exons were
sequenced on an ABI Prism 3100 capillary sequencer
(Applied Biosystems, Foster City, CA, USA) and the data
Madison, WI, USA). BsrBI and Tsp509I restriction enzymes
were used to screen 646 control chromosomes for A249E
and K424R mutations, respectively, whereas 366 control
chromosomes were screened with the following enzymes:
HpyCH4V (Q253L and P327H), SphI, (G42V), BsrI (Q119R)
and TaqaI (D216G). Digestion products were scored following
electrophoresis on a 1% agarose gel with ethidium bromide.
4.5 software (GeneCodes,
Quantitative polymerase chain reaction and array CGH
qPCR was used to screen a subset of 32 patients for copy
number alterations of GDF6 (Taqman, Applied Biosystems).
Primers were designed using Primer Express software (ABI)
(available on request) and qPCR was performed together
with 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 for
1 min (ABI Prism, 7000, Sequence Detection System). A
DNA sample from the proband of the SHFM pedigree that
maps to 8q21.11-q22.3 was hybridized to an oligonucleotide
array comprising 385,000 probes at a mean spacing of 6-kb
as described elsewhere (46).
Reporter gene assay
After adding a Kozak consensus sequence to permit ribosomal
binding on wild-type, A249E and K424R human GDF6
cDNA, sequences were initially cloned into TOPO4 (Invitro-
gen), then CS2 vector and 1–200 pg of capped, poly-A tailed
mRNA (mMessage mMachinew, Ambion Inc, Austin, TX,
USA) were injected into 1-cell embryos. For the biochemical
assay, PLM cells were harvested from embryonic age (E) 11.5
CD-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-well
plates. Briefly, DNA-transfection mixtures were aliquoted into
wells followed by the addition of approximately 100,000 PLM
cells, and wells were topped-up to a total volume of 100 ml.
Media was replenished 24 h post-transfection, and lysates
were collected 48 h post-transfection. Luciferase activity was
measured using the Dual Luciferase Kit (Promega, Madison,
WI, USA) and firefly luciferase was normalized to an internal
Renilla luciferase control. Luciferase assays were performed in
quadruplicate and repeated three times.
Western blot analysis
The coding sequence of GDF6 and mutations A249E and
K424R (minus the stop codon) were incorporated into
V5-tagged Destination vector (Invitrogen, Carlsbad, CA,
USA), and confirmed by sequencing. Transient transfections in
COS7 cells were performed with FuGENE (Roche, Diagnostics,
Indianapolis, IN, USA) according to manufacturer’s instructions
on 100 mm plates with culture media and lysates collected 48 h
post-transfection as previously described (93). Proteins were
extracted, separated on a 15% SDS–PAGE gel and transferred
to nitrocellulose membranes (BioRad, Hercules, CA, USA),
which were incubated with anti-V5 (1:10,000), secreted alkaline
phosphatase (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 by
chemiluminescence (Pierce, ThermoScientific, Rockford, IL,
USA). b-Galactosidase was co-transfected with GDF6þV5
vectors and examined with a b-galactosidase enzyme assay
(Promega) for transfection control.
Zebrafish phenotyping and in situ hybridization
Zebrafish (AB strain) knockdown experiments were performed
as described earlier (2), with an additional gdf6a morpholino
containing five nucleotide substitutions as a control (sequences
available on request). RNA whole-mount in situ hybridizations
for myod, her7, unc45, nog1, nog2, gremlin1 and gdf5 were
performed as previously described (94). Stained embryos
were mounted in 70% glycerol and photographed with a
camera. For qPCR validation of in situ hybridizations,
1118 Human Molecular Genetics, 2009, Vol. 18, No. 6
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 and
noggin3 using the ‘Roche Applied Science Universal Probe
index.jsp (available on request). Expression levels were
quantified by qPCR using SYBR Green chemistry (Stratagene),
where ef1a was used as a control. All PCRs were performed
twice in triplicate.
Murine genotyping and phenotyping
Gdf6þ/2mice [strain Gdf6tm1Lex(MGI:3604391) http://www.
informatics.jax.org] were housed and handled in accordance
with the University of Alberta Animal Policy and Welfare
Committee protocols. Mice were genotyped using allele-
specific primers (sequences and conditions available on
request) on DNA derived from ear-notched tissue. Mice
were anaesthetized using isoflurane and pupils were dilated
with Tropicamide (Alcon, USA). Images were initially captured
with a digital camera through an OPMIwVISU 160 microscope
(Zeiss, Germany). Subsequent examinations were undertaken
using an endoscope with an attached otoscope (1218AA;
Karl Storz, Tuttlingen, Germany) (95) and still images and
videos were captured on a Telecam SLII camera (reference
202130-20, 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 mm3voxels,
from the 1808 angular rotational scans (0.58 incremental
steps) and reconstructed using a modified Feldkamp algorithm
(40). All image data were Gaussian-filtered and globally
thresholded using standardized minimum and maximum
cross-section to image conversion values of 0.0–0.0600,
respectively, to extract the mineralized phase representing
the three-dimensional (3D) bone architecture. Qualitative
assessment of skeletal formation was performed from rendered
visualizations of the 3D architecture, while quantitative analysis
of the vertebral bodies was undertaken with morphometrical
analysis software (CTan, Skyscan NV, Aartselaar, Belgium)
for the bone volume ratio [volume of bone/total volume of
bone and soft tissue], as described elsewhere (41).
The authors thank the patients for participating in this study;
Dr Fred Berry for very helpful discussions; Drs Karen
Temple, David Fitzpatrick and Raymond Clarke for provision
of DNA samples; and Anthony Lott, Helen Chung, Mathew
Laroque, May Yu, Tim Footz, Yoko Ito, Hermina Strungau,
Timothy Erickson, B. Hemadevi and B. Suganthalakshmi,
Drs Stacey Bleoo, Martin Somerville and Gino Fallone for
Conflicts of Interest statement. None declared.
The Canadian Institutes of Health Research (to O.J.L. and
T.M.U.), Alberta Heritage Foundation for Medical Research
and Canadian Foundation for Innovation (to O.J.L.), National
Scientific Engineering Council (to C.R.F. and A.J.W.) and
Alberta Ingenuity Fund (to A.J.W.). A.J.W. and O.J.L. are
recipients of Canada Research Chairs.
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