HUMANMUTATION 29(8),1017^1027,2008

RESEARCH ARTICLE

Mutations in GDF6 Are Associated With VertebralSegmentation Defects in Klippel-Feil Syndrome

May Tassabehji,1 Zhi Ming Fang,2 Emma N. Hilton,1,3 Julie McGaughran,4 Zhongming Zhao,5

Charles E. de Bock,2 Emma Howard,6 Michael Malass,1 Dian Donnai,1 Ashish Diwan,7

Forbes D.C. Manson,3 Dedee Murrell,7 and Raymond A. Clarke2,7,8�

1Academic Unit of Medical Genetics and Regional Genetics Service, University of Manchester, St Mary’s Hospital, Manchester, United Kingdom;2Human Molecular Genetics Laboratory, University of North South Wales, Clinical School of Medicine, St George Hospital, Sydney, Australia;3Centre for Molecular Medicine, University of Manchester, Manchester, United Kingdom; 4Genetic Health Queensland, Royal Children’s Hospitaland Health District, Herston, Brisbane, Queensland, Australia; 5Department of Psychiatry and Human Genetics and Center for the Study ofBiological Complexity, Virginia Commonwealth University, Richmond, Virginia; 6Regional Genetics Service, St. Mary’s Hospital, Manchester,United Kingdom; 7Orthopaedic Research Institute, University of North South Wales, St George Hospital, Kogarah, North South Wales, Australia;8Cancer Care Centre, St George Hospital, Kogarah, North South Wales, Australia

Communicated by Stylianos E. Antonarakis

Klippel-Feil syndrome (KFS) is a congenital disorder of spinal segmentation distinguished by the bony fusion ofanterior/cervical vertebrae. Scoliosis, mirror movements, otolaryngological, kidney, ocular, cranial, limb, and/ordigit anomalies are often associated. Here we report mutations at the GDF6 gene locus in familial and sporadiccases of KFS including the recurrent missense mutation of an extremely conserved residue c.866T4C(p.Leu289Pro) in association with mirror movements and an inversion breakpoint downstream of the gene inassociation with carpal, tarsal, and vertebral fusions. GDF6 is expressed at the boundaries of the developingcarpals, tarsals, and vertebrae and within the adult vertebral disc. GDF6 knockout mice are best distinguishedby fusion of carpals and tarsals and GDF6 knockdown in Xenopus results in a high incidence of anterior axialdefects consistent with a role for GDF6 in the etiology, diversity, and variability of KFS. Hum Mutat29(8),1017–1027, 2008. rr 2008 Wiley-Liss, Inc.

KEY WORDS: Klippel-Feil; KFS; GDF6; vertebral fusion; development; etiology

INTRODUCTION

Klippel-Feil syndrome (KFS; MIM]s 214300, 118100, 148900)is a complex skeletal disorder characterized by congenital fusion ofvertebrae within the anterior/cervical spine [Clarke et al., 1998;Gunderson et al., 1967; Klippel and Feil, 1975]. Vertebral fusionappears to be caused by a failure in the normal segmentation ofvertebrae during the early weeks of fetal development anddefective somitogenesis has been postulated as a mitigating factor[Clarke et al., 1998; Gunderson et al., 1967]; however, theetiology of KFS is still unknown and no definitive disease-causinggenes have yet been identified [McGaughran et al., 2003].

Both sporadic and familial (autosomal dominant) forms of thesyndrome display clinical heterogeneity [Clarke et al., 1995] withvariable degrees of vertebral fusion (Types I–III), spinal instabilityand associated disc degeneration, cranial and orofacial anomaliesincluding cleft palate, facial dysmorphism, sensorineural, andconductive hearing impairment, vocal impairment, carpal anoma-lies, appendicular hypoplasia, Sprengel deformity (MIM] 184400),and short neck with low hair line, as well as occasional broadspectrum anomalies of the heart, kidney, genitourinary system, eye,including Wildervanck syndrome (MIM] 314600) [Gupte et al.,1992], mirror movements, and other neurological anomalies[Cincinnati et al., 2000; Clarke et al., 1998]. Some phenotypiccomponents of KFS overlap with the combination of Mullerianduct aplasia, unilateral renal aplasia, and cervicothoracic somite

dysplasia (MURCS Association; MIM] 601076) [Strubbe et al.,1992] and Jarcho-Levin syndrome (MIM] 277300) [Martinez-Frias et al., 1998].

KFS appears to be a multifactorial syndrome in which most casesmanifest either variable expressivity and/or unique associatedanomalies [Clarke et al., 1998]. A number of chromosomeabnormalities have also been associated with KFS, providingcandidate loci, for example, a translocation t(5;17)(q11.2;q23) in asporadic KFS patient with associated brachydactyly [Fukushima et al.,

Published online 18 April 2008 in Wiley InterScience (www.interscience.wiley.com).

DOI10.1002/humu.20741

The Supplementary Material referred to in this article can be ac-cessed at http://www.interscience.wiley.com/jpages/1059-7794/suppmat.

Received 2 October 2007; accepted revised manuscript 3 January2008.

Grant sponsors: Garnett Passe and Rodney Williams MemorialFoundation; National Health andMedical Research Council of Austra-lia; Scoliosis Research Society; Grant sponsor:WellcomeTrust; Grantnumber: 061183.

MayTassabehji and Zhi Ming Fang are joint ¢rst authors.

�Correspondence to: Dr. Raymond A. Clarke, Human MolecularGenetics, University of New SouthWales, Research & Education Bldg,St George Hospital, South St, Kogarah, NSW 2217, Australia.E-mail: r.clarke@unsw.edu.au

rr 2008 WILEY-LISS, INC.

1995], and an inversion involving chromosome 2, inv(2)(p12q34) ina KFS patient with hypodontia [Papagrigorakis et al., 2003]. The fewreported autosomal dominant families display variable anomaliesincluding os odontoideum [Morgan et al., 1989], basilar impression[Gunderson et al., 1967], omovertebral bone [Larson et al., 2001],cleft palate [Thompson et al., 1998], Dubowitz syndrome [Takahiraet al., 2005], macrocephaly, and in one family with microcephaly atranslocation t(5;8)(q35.1;p21.1) locus was found segregating withthe syndrome [Goto et al., 2006]. These characteristics and theabsence of a consistent genomic locus indicate that KFS is geneticallyheterogeneous [Clarke et al., 1998].

We previously proposed chromosome 8q as a candidate locusbased on a paracentric inversion inv(8)(q22.2q23.3) foundsegregating with anterior/cervical fusions in a large autosomaldominant KFS family (Family KF2-01) [Clarke et al., 1995]. Herewe localize both of the inversion breakpoints on 8q. The proximalbreakpoint is located 623 kb downstream of GDF6 (MIM]601147), in a genomic region known to harbor GDF6 long-rangeenhancer elements [Mortlock et al., 2003; Settle et al., 2003].Subsequent mutation screening of a large and clinically diverseKFS cohort has identified GDF6 missense mutations in bothfamilial and sporadic KFS patients. Gdf6 knockdown experimentsin Xenopus laevis resulted in anterior axial defects consistent with arole for GDF6 in KFS.

MATERIALSANDMETHODSSubjects

Ethically approved informed consent was obtained from allparticipants in this study. Fresh blood samples were collected usingstandard protocols for use in fluorescent in situ hybridization(FISH) and DNA analyses.

FISHChromosome Inversion Analysis

Karyotyping of Family KF2-01 revealed inversion breakpoints at8q22.2 and 8q23.3 [Clarke et al., 1995]. M-FISH was undertakenusing region-specific and partially overlapping chromosome BACprobes (Invitrogen, Mount Waverley, Australia) selected fromgenomic maps listed on the National Center for BiotechnologyInformation (NCBI) database (www.ncbi.nih.gov). Metaphasespreads were prepared from PHA-stimulated lymphocytes, cul-tured at 371C for 72 hr. BAC DNA was nick-translated usingfluorescent labeled dUTP (spectrum green and spectrum red;Vysis, Inc., Abbots Laboratory, Downers Grove, IL). Hybridizationto metaphase chromosomes was performed using a standardprotocol [Pinkel et al., 1986]. A total of 400 ng of labeled DNAwas used per slide and repetitive sequences blocked by annealingwith a 400-fold excess of Cot-DNA (Immunodiagnostics Pty Ltd,Woburn, MA) at 371C for 45 min, prior to hybridization.Chromosomes were counterstained with DAPI. A total of 50metaphases were analyzed for each hybridization. Images werecaptured and merged using an Imstar digital FISH imaging system(Immunodiagnostics).

Inversion Breakpoint Analysis

A total of two BAC clones (AC026561 and AC012238) fromboth ends of the inversion gave split signals on M-FISH. Multipleprimer pairs were designed from their genomic sequences to amplifythe junction fragments that spanned the inversion breakpoints. Anarray of forward PCR primers at 5-kb intervals across both breakpointregions were designed to amplify the proximal inversion breakpoint;forward primers from both breakpoint regions were mixed together ina single PCR reaction with genomic DNA from members of Family

KF2-01. Primers used to amplify the proximal inversion breakpointwere: 1F primer 50-ATCCCTTAGTTGAACACAAAAAGCA-CAAGC-30 (from BAC AC026561) and the 2F primer 50-TTCTATAAAGATCATCCATGCTAAACACTG-30 (from BACAC012238). To amplify the distal inversion breakpoint this PCRstrategy was repeated using a mixed set of reverse primers: 1R primer50-TGTATGAGAGTTTTGGTGGTTCCACATC-30, and 2R 50-GATAAGGACTGAGATATGCCCTGGT-30. Long-range break-point PCR was performed in a 25ml reaction containing 50 ng ofgenomic DNA, 0.2mM of each primer, 200mM dNTPs, and 1 UElongase enzyme (Invitrogen). Cycling conditions were: an initial 3-min denaturation step at 951C; 32 cycles of denaturation at 951C for30 s, annealing at 601C for 30 s, extension at 721C for 7 min;followed by a final extension at 681C for 3 min.

DNA Sequencing

PCR products were purified using the QIAquick Spin PCRpurification kit (Qiagen, Doncaster, Australia). DNA sequencingwas performed using the ABI Big Dye Terminator version 3.1 cyclesequencing kit (ABI, Foster City, CA) according to the manufac-turer’s instructions (i.e., 5 ng [5ml]) purified PCR amplicon, 4mlreaction premix, 2ml 5� sequencing buffer, 3.2 pmol (2ml)appropriate primer, and 7ml deionized water were added in a 96-well microtiter plate. Cycling conditions were: 961C for 1 min; 25cycles at 961C for 10 s, 501C for 5 s, and 601C for 4 min. Sequencingproducts were purified using ABI Centri-Sep spin columns andresuspended samples resolved on an ABI 377 DNA sequencer,according to the manufacturer’s instructions. Sequences wereanalyzed using the BioEdit biological sequence alignment editor(v 5.0.9.1; Tom Hall, Isis Pharmaceuticals, Carlsbad, CA).

Mutation Screening

The two exons of GDF6, including at least 50 bp of flankingintronic sequence (GenBank accession number AJ537424), werescreened in our patient cohort by automated sequencing. GDF6primers (synthesized by Invitrogen Australia) were designed usingthe Primer3 program (http://frodo.wi.mit.edu) (Supplementary TableS1; available online at http://www.interscience.wiley.com/jpages/1059-7794/suppmat). PCR reactions of 25ml were prepared (50 nggenomic DNA, 0.2mM of each primer, 200mM dNTPs in 1� PCRbuffer with 5% DMSO and 0.25 U Taq polymerase [Promega,Annandale, Australia]) and cycled using the following conditions:denaturation at 951C for 4 min followed by five touchdown cycles ofdenaturation at 951C for 40 s, annealing at 651C for 40 s, andextension at 721C for 50 s; then 28 cycles of 951C for 40 s, 601C for40 s, and 721C for 50 s, with a final extension for 10 min at 721C(CG1-96; Corbett Research, Mortlake, Australia). PCR productswere resolved by electrophoresis in 1.4% agarose gels and purifiedusing the Promega Wizard gel purification system before bidirec-tional sequencing. All mutations and polymorphisms were confirmedfrom a second PCR product amplification. DNA mutationnumbering is based on the cDNA sequence, where11 correspondsto the A of the ATG translation initiation codon in RefSeqNM_001001557.1. The initiation codon is counted as codon 1.

Protein SequenceAlignment

Sequence alignments were carried out using ClustalW (www.ebi.ac.uk/clustalw).

Proteins from aligned species included Homo sapiens (gi: accessionnumber Q6KF10), Macaca mulatta (gi: XP_001090825), Musmusculus (gi: P43028), Ratus norvegicus (gi: Q6HA10), Xenopus

1018 HUMANMUTATION 29(8),1017^1027,2008

laevis (gi:Q9W753), Danio rerio (gi: Q12938), and Tetraodonnigroviridis (gi: Q4SSW6).

ComparativeAnalysis of Conserved NoncodingSequences (CNSs)

In view of the proximity of the KF2-01 inversion breakpoint tothe GDF6 gene, and previous studies that demonstrate GDF6enhancer elements downstream of the gene, we performedcomparative analysis of genomic sequences from multiple species.Genomic sequences from the proximal breakpoint region betweenGDF6 (623 kb 50 of the breakpoint) and C8orf37 (hypotheticalprotein LOC157657) (194 kb 30 of the breakpoint) were extractedfrom the Ensembl and NCBI GenBank databases (www.ensem-bl.org, version 41.36c; www.ncbi.nlm.nih.gov, build 36) for human,chimpanzee, dog, mouse, rat, chicken, and opossum and analyzedfor any conserved noncoding sequences (CNSs) using VISTA(http://genome.lbl.gov/vista) with the human sequence as thereference sequence [Frazer et al., 2004]. Strict CNS criteria weredefined to be Z100 bp ungapped alignment with at least 88%identity. The human gene annotation was obtained from theEnsembl database and the repeat information was obtained fromRepeatMasker (www.repeatmasker.org).

Xenopus Microinjection of Morpholinos

Xenopus laevis embryos were obtained and prepared for injectionby standard methods [Hanel and Hensey, 2006]. Morpholinoinjections were performed at one-cell stage using a Picospritzer IIImicroinjector (Intracel, Royston, United Kingdom) to inject up to22-nl volumes. During injection and for subsequent incubation,embryos were maintained in 0.4�MMR/6% Ficoll/1� gentamycin at 141C. Prior to gastrulation, embryos were

removed to 0.1� l MMR/1 � gentamycin at 14 1C for theremainder of culture. A morpholino targeted to the ATG startcodon of X. laevis Gdf6 (RefSeq NM_001090364.1) and astandard control morpholino were kind gifts of C. Hensey [Haneland Hensey, 2006]. The sequence of the Gdf6 morpholino was 50-GCAGAGGGCTCCTGTATGTATCCAT-30.

RESULTSClinical Phenotypes of Familial Cases

Detailed clinical investigations of two KFS families (KF2-01 andKF2-03) revealed C2-3 vertebral fusion (Fig. 1A–C) in all 24affected family members radiographed. This and the diminishingfrequency of more caudal fusions within both families is indicativeof the autosomal dominant KF2 class recently described in aclassification update [Clarke et al., 1998]. The extent of fusionvaried considerably both within and between families, rangingfrom a narrow fusion-bridge between C2-3 in some individuals(Fig. 1A) to multiple block fusions in others (Fig. 1B and C)(thinning of the C2-3 vertebral interspace and minimal fusionbridges in KFS individuals often go unreported during generalradiological assessment). Fusion occurred between vertebral bodiesand laminae and rarely between spinous processes. The height offused vertebral bodies was usually maintained (Fig. 1A and B) withoccasional vertebral malformation and disc degeneration (Fig. 1C).Scoliosis was evident in regions of the spine devoid of vertebralfusion. In Family KF2-01, bilateral fusion of the carpals and tarsalswas evident including fusion between the triquetrum and lunate,hamate and capitate, and pisiform and hamate (Fig. 1D–F) similarto the phenotype of GDF6 knockout mice. Affected members ofthis family manifest restricted flexibility of the hands, wrist, elbow,feet, and legs. Both families manifest varying degrees of negative

FIGURE 1. Clinical features of KFS patients. A: Lateral view of the KFS cervical spine.The arrow indicates the minimal C2-3 fusion-bridge observed in some a¡ected individuals from Families KF2-01 and KF2-03. B: Skipped vertebral fusions observed within thecervical spineof ana¡ected individual fromFamilyKF2-01 [Clarkeet al.,1996]. (C2-3 fusion is alwayspresent ina¡ected individualsfromFamiliesKF2-01andKF2-03).C: Block fusionpattern (C2-3 andC5-6) in theFamilyKF2-03 proband.There is signi¢cant discdegeneration at the C4-5 interspace. D: Dorsal radiograph of the wrist showing fusion/coalition between the hamate (H) and capi-tate (C), the lunate (L), and triquetrum (T) and in a lower plane between thehamate andpisiform (P) (FamilyKF2-01, Patient IV-10).E: Lateral view of hamate-pisiform coalition (Family KF2-01, Patient IV-10). F:Coalition between ¢rst and second tarsals and adja-centmetatarsals inFamilyKF2-01.G:An18-week-old female fetus, PatientKFS-66,withmarkedshorteningandspinal angulation inthe thoracic region.Theneckwasshortenedwith signi¢cant nuchaledema.Therewas ¢fth ¢ngerclinodactylywith rocker-bottom feetand prominent heels. H: Patient KFS-66 X-rays showmultiple segmentation abnormalities a¡ecting the spine. Note thewidening ofthe interpedicular distance in the cervical and upper thoracic spine together with hemi- and butter£y vertebrae.The rib cage is shortand there is amarked failure of development of the lumbar and sacral spine segments. [Color ¢gure can beviewed in the online issue,which is available at www.interscience.wiley.com.]

HUMAN MUTATION 29(8),1017^1027,2008 1019

ulna variance. Family KF2-01 also presented with additionalskeletal anomalies (listed in Table 1) including otolaryngealcartilage malformations associated with moderate hearing impair-ment and severe vocal impairment [Clarke et al., 1994]. Vocalimpairment always segregated with vertebral fusion in Family KF2-01. Two affected members of Family KF2-03 presented withprominent flaring of the lower ribs, another manifest congenitalomphalocele (due to a failure in ventral body wall closure), andone suffered painful disc degeneration at the site of a severe axialkink within the cervical spine.

Clinical Phenotypes of Sporadic Cases

A total of two sporadic cases of KFS (Patients KFS-23 and KFS-66) presented with broad spectrum KFS phenotypes consistentwith the KF1 class described in the updated classification [Clarkeet al., 1998]. Patient KFS-23 was an adult male with a classicalKF1-like phenotype including a short neck, mirror movements,relative macrocephaly (497th), a squint as a child (not requiringoptical glasses), and left Sprengel anomaly of the shoulder.Cytogenetic analysis revealed a normal male karyotype. PatientKFS-66 was a female fetus assessed following termination ofpregnancy for antenatal diagnosis of multiple segmentationabnormalities affecting the entire spine and ribs and hemi- andbutterfly-vertebrae (Fig. 1G and H). The spinal cord was flattenedand dysplastic and ended at the mid-thoracic region. There wasalso developmental failure of the lumbar and sacral spine segments,fifth finger clinodactyly and rocker-bottom feet, Arnold Chiari typeII malformation, moderate dilatation of the lateral ventricles,absent right kidney and uterus alongside anal atresia, indicatingphenotypic overlap with Jarcho-Levin and Cassamassima-MortonNance syndromes (MIM] 271520). Cytogenetic analysis revealeda normal female karyotype and we detected no mutations in DLL3,MESP2, or LFNG (genes linked to Jarcho-Levin, spondylocostaldysostosis, and other vertebral phenotypes) [Sparrow et al., 2006].

Inversion Breakpoint LocalizedWithin GDF6 Locus

To refine the location of the inversion (inv(8)(q22.2q23.3)[Clarke et al., 1995] breakpoints within the now expanded fivegeneration autosomal dominant Family KF2-01 (Fig. 2A), weperformed M-FISH chromosome analysis using chromosome8–specific BAC probes. BAC clones (AC026561 and AC012238)spanned the inversion junctions and gave split hybridization signals(Fig. 2B). Multiple PCR primer pairs designed from each BACallowed unique breakpoint junction PCR fragments to be amplifiedin all 19 of the affected members tested, verifying cosegregation ofthe inversion with the disease phenotype (data not shown).Sequencing of the breakpoint PCR amplicons identified theproximal and distal inversion breakpoints on chromosome 8q atnucleotide positions 96544749 and 116078713, respectively (NCBIbuild 36) (Fig. 2C). The inverted segment was 19,533,963 bp andno DNA loss was evident at either breakpoint and no coding genesappear to be directly disrupted (Fig. 3).

The distal breakpoint occurs within an intergenic region 1.6 Mb50 from CSMD3 and 400 kb 30 from TRPS1 (Fig. 3). CSMD3 isexpressed predominantly in fetal brain [Shimizu et al., 2003], andTRPS1 mutations cause trichorhinophalangeal syndrome type I(MIM] 190350). TRPS1 patients harboring deletions across thisregion 30 of the TRPS1 gene do not present with KFS symptomsbut rather present with abnormalities of the phalanges, slowgrowing and sparse scalp hair, medially thick and laterally thineyebrows, bulbous tip of the nose, long flat philtrum, thin upper lipwith vermilion border, and protruding ears [Ludecke et al., 2001].

FIGURE 2. Molecular analysis of Family KF2-01. A: FamilyKF2-01 pedigree with inv(8)(q22.2q23.3). Question marksdenote those individualswithnoobviousKFS-associatedpheno-type, not available for analyses. A¡ected members manifestC2-3 vertebral fusion, varying degree of vocal impairment andsome degree of pisiform expansion. Approximately 50% ofa¡ected members manifest carpal fusion ando50% haveother associated anomalies (Table 1). B: FISH analysisshows £uorescent BAC clones AC026561 (red) and AC012238(green) spanning the 8q22.2 and 8q23.3 breakpoints respec-tively; both yield split hybridization signals in a¡ectedmembers.C: Chromatograms displaying the nucleotide sequenceacross the proximal (higher) and distal (lower) inversion break-points. Shaded region represents the uninterrupted nucleotidesequence across the proximal inversion breakpoint (position96544749 on 8q22.2; 623 kb downstream of GDF6) (NCBIbuild 36).

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As such, the TRPS1 gene appears to have had no obviousinfluence on the Family KF2-01 phenotype.

The proximal Family KF2-01 inversion breakpoint located623 kb 30 of GDF6 appears to be largely responsible for KFS in thisfamily. GDF6 is a member of the BMP family of secreted signalingmolecules implicated in joint and vertebral formation [Mortlocket al., 2003; Settle et al., 2003]. The genomic region betweenGDF6 and LOC157657 is known to harbor GDF6 long-rangeenhancer elements [Mortlock et al., 2003] and we identified 23extremely conserved CNSs on both sides of the proximalbreakpoint (Fig. 3B). The anatomical sites of all fusions (vertebral,

carpal, and tarsal) and associated skeletal anomalies within FamilyKF2-01 (Table 1) precisely correspond with the discrete andspatially-restricted sites of Gdf6 expression during mouse devel-opment [Mortlock et al., 2003; Settle et al., 2003] and the sites offusions in Gdf6 knockout mice. These previous studies provideevidence for GDF6 involvement in the segmentation of carpal/tarsal precursors leading to joint formation and in the normaldevelopment of vertebral bodies, and articular and spinouscartilage [Mortlock et al., 2003; Settle et al., 2003]. With theexception of GDF6, no other gene located adjacent to the FamilyKF2-01 inversion breakpoints has recognized roles or expression

FIGURE 3. CharacterizationofFamilyKF2-01inversionbreakpoint. A: Schematic of the genomic regionspanning theFamilyKF2-01inversion breakpoints.The Family KF2-01 breakpoint is located 623 kb 30 ofGDF6 and 194 kb 50 of C8orf37 (hypothetical proteinLOC157657). Arrows represent genes within the breakpoint region. Breakpoint BACs are shown as solid bars and the two previouslylocalizedGDF6 tissue-specifying enhancer elements (LE, laryngeal enhancer and DJE, distal joint enhancer) [Mortlock et al., 2003]are shown as cross-hatched bars. Arrows represent genes within the breakpoint region.The full-length GDF6 protein monomer isdepicted belowwith signal peptide (black), prodomain (light gray), and active/mature signaling domain (dark gray); themature sig-naling domain contains seven cysteine residues that are conserved amongTGFband BMP family members.Vertical arrows mark therelative position of the missense mutations p.Ala249Glu and p.Leu289Pro within the prodomain. B:VISTA plot displaying evolu-tionary conserved sequences in the breakpoint region. CNS (in red) with Z100 bp ungapped alignment and Z87% identity. A totalof 23 CNSs spanning the intergenic region between GDF6 and LOC157657 that satis¢ed the de¢nition for all alignments betweenhuman and the other ¢ve species (chimpanzee, dog, mouse, rat, and opossum) aremarked.

HUMAN MUTATION 29(8),1017^1027,2008 1021

patterns which overlap adequately to explain the Family KF2-01familial phenotype. As such, GDF6 was screened for mutations ina large KFS cohort representing a broad diversity of KFSphenotypes (KF1–4) [Clarke et al., 1998], many with uniqueanomalies (no GDF6 mutations were detected in Family KF2-01).

GDF6 Missense Mutations in KFS Patients

Direct nucleotide sequencing of GDF6 (two coding exons andflanking splice sites) in 127 KFS individuals (six familial probandsand 121 sporadic individuals) led to the identification of a recurrentmissense mutation in two sporadic cases and a second missensemutation segregating with a familial form of the syndrome. In threegenerations of the autosomal dominant Family KF2-03 thep.Ala249Glu (c.746C4A) mutation segregates strictly with theKFS phenotype (Fig. 4) and the recurrent mutation p.Leu289Pro(c.866T4C) was identified in two unrelated sporadic cases(Patients KFS-23 and KFS-66). These mutations were not detectedin 708 control chromosomes tested from ethnically matchedindividuals, giving495% power to distinguish a normal sequencevariant from a mutation [Collins and Schwartz, 2002]. None of thebase substitutions found was present in the NCBI dbSNP database.Many patients without GDF6 mutations also showed no evidence ofmicrodeletions (data not shown). A total of two new polymorphismswere identified in�4% of both the KFS and control populations:c.406128C4A and c.936G4C (p.Ser312Ser) (data not shown).

Recurrent GDF6L289P MissenseVariant

The recurrent heterozygous missense mutation in exon 2(c.866T4C), detected in two unrelated sporadic cases, involvesthe substitution of leucine for proline at position 289 (p.Leu289Pro)

(Fig. 4B). The structural and functional consequences of thismutation are not yet clear since the 3D crystal structure of thisportion of the protein is unknown; however, the properties ofleucine and proline residues differ considerably. Leucine is analiphatic, hydrophobic amino acid that is generally buried in foldedproteins whereas proline is a cyclic aliphatic amino acid oftenfound at the end of a helix or in turns or loops. Unlike other aminoacids that exist almost exclusively in the trans-form in polypep-tides, proline can exist in the cis-configuration in peptides. The cisand trans forms are nearly isoenergetic and cis/trans isomerizationcan play an important role in the folding of proteins. Leucine 289is located 47 amino acids from the C-terminal cleavage site of theGDF6 prodomain and is conserved between all species tested andbetween all human GDFs (Fig. 4C).

GDF6A249E MissenseVariant

The heterozygous missense mutation in exon 2, c.746C4A(Fig. 4A), segregates with vertebral fusions (always inclusive of theC2-3 fusion) [Clarke et al., 1998] in all three generations of FamilyKF2-03 (Fig. 4A). The c.746C4A mutation resulted in thesubstitution of glutamic acid for alanine 249 also within the C-terminal-half of the GDF6 prodomain (p.Ala249Glu). Alanine is asmall nonpolar molecule while glutamic acid is polar and usuallyfound on the outside of proteins, where it is free to interact withthe aqueous intracellular surroundings. Alanine 249 is conservedbetween humans and mice, but not canine, frog, and fish species,and is also conserved in two of the three human GDFs (Fig. 4C).A similar charge variant p.Ser204Arg in the prodomain of GDF5in a family with brachydactyly Type C [Everman et al., 2002]shows similar conservation (Fig. 4C). It appears that the properties

TABLE 1. Correspondence BetweenDiscreteGdf6 Expression Patterns andCartilage, Bone, Ligament, andJoint anomalies of Family KF2-01

Gdf6 Expression inmouse embryos Human Family KF2-01

Cartilage/ligamentstructure

Endogenous[Settle et al.,

2003]

LacZ[Mortlocket al., 2003]

Gdf6�/�[Settle et al.,

2003] Phenotype (% a¡ected)

Vertebralinterspaces

Vertebral joints Vertebral joints Interspace cartilagereduced; scoliosis

Fusion of vertebral bodies and lamina (100); scoliosis in thoracic andlumbar spine (100)

Dorsal neuraltube

Craniocaudalgradient

Craniocaudal fusion frequency gradient

EarPinnacartilage

Pinna cartilage Absent pinna cartilage or ears low set (90)

Meatuscartilage

Facial cartilage Restricted auditory canal and/ormicrotia (80)

Middle earjoints

Middle ear joints Middle earjoints

Middle ear jointsreduced

Conductive hearing impairment (10)

LarynxCartilages Laryngeal

cartilagesThyroid and

cricoidcartilage

Malformed thyroid and cricoid cartilages; infantile epiglottis and narrowglottic chink

Vocal folds Vocal folds Vocal folds Completely aphonic; no fold vibration; one female; shortmisaligned vocalfolds; severe vocal impairment (80); learning di⁄culties (50)

Palate Palate Palate High palateFace andmouth

Teeth Teeth Microstomia, micrognathia and/or short tongue (80)

Carpal joints Carpal joints Carpal joints Bilateral carpalcoalition

Bilateral carpal coalition (50); gradation of pisiform expansion (100)

Tarsal joints Tarsal joints Tarsal joints Tarsal coalition Bilateral tarsal coalitionTalus joint Talus joint Talus coalition Talocalcaneal coalitionTendons of feetand hands

Di⁄culty grasping, writing, and toe-walking (100)

Elbow joint Elbow joint Elbow joint Negative ulnavariance

Restricted elbowmovement (100); negative ulna variance

Hip joint Hip joint Hip joint Perthes; onemale a¡ected family memberScapula Edge of scapula Elevated scapula; Sprengel anomaly (50)

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rather than the specificity of this residue may be functionally moreimportant within the prodomain.

Phenotypes AssociatedWith Gdf6 Knockdown inXenopus laevis

To further define the pathological consequence of disruptingGDF6 dosage, we recapitulated a proven morpholino (Gdf6 MO)protocol employed to investigate the role of Gdf6 in ocular

development in Xenopus laevis [Hanel and Hensey, 2006]. NormalGdf6 expression increases significantly around the mid-gastrulastages until the tailbud stages and its reduction leads to malformedembryos. In contrast to the control morpholino, which elicited novisible abnormal phenotype, the Gdf6 MO induced axial andocular defects in a dose-dependent fashion when injected at thesingle-cell stage (Fig. 5; Table 2). Axial defects were seen in 21%and 39% of embryos at 10 ng and 20 ng doses, respectively, inaddition to and independently of eye defects [Hanel and Hensey,

FIGURE 4. Molecular ¢ndings in KFS. A: Family KF2-03 pedigree. Chromatogram of c.746C4A (p.Ala249Glu) GDF6 missensemutation (RefSeq NM_001001557.1) segregating with vertebral fusion (upper) and normal una¡ected family members (lower). B:Recurrent sporadic missense mutation c.866T4C (p.Leu289Pro) in two unrelated sporadic KFS patients (Patients KFS-23 andKFS-66).C:ClustalWGDFprotein alignments. Mutated residues (boxed) ofGDF6 (KFS) andGDF5 (brachydactylyTypeC) [Evermanet al.,2002]. Phylogenic alignment around: (i) GDF6 alanine 249; (ii) leucine 289; and (iii) GDF family alignment of C-terminal sec-tion of prodomain. [Color ¢gure can be viewed in the online issue,which is available at www.interscience.wiley.com.]

HUMANMUTATION 29(8),1017^1027,2008 1023

2006]. The most prevalent phenotype of abnormal anterior axialalignments resulted in embryos with head positions deviating inthe vertical and lateral plane (Fig. 5C [v–vi]). A total of 14% ofmorphants manifest both axial and ocular defects; however, rescueof either phenotype was not possible due to the strong ventralizingeffects of exogenous overexpression of GDF6 during development[Hanel and Hensey, 2006].

DISCUSSION

BMPs are important regulators of chondrogenesis and skeleto-genesis and the GDFs (5/6/7) represent a distinct vertebratespecific subgroup within the BMP family that regulate the normalformation of skeletal joints in the limbs, skull, and axial skeleton[Settle et al., 2003]. GDF6 is expressed within developing jointsincluding the developing vertebral interspace, and knockoutmouse studies provide proof of GDF6’s role in joint fusion[Mortlock et al., 2003; Settle et al., 2003]. This is the first reportlinking GDF6 with joint fusion and KFS in humans. The initialindication of a link between GDF6 and KFS came from aparacentric inversion (inv(8)(q22.2q23.3)) breakpoint 623 kbdownstream of GDF6, which we found segregating with vertebralfusion and other skeletal anomalies (Table 2) in the autosomal

dominant Family KF2-01 [Clarke et al., 1995]. Gdf6 knockoutmice are best distinguished by unique patterns of carpal and tarsalfusions [Mortlock et al., 2003; Settle et al., 2003] and acomparable pattern of carpal and tarsal fusions was foundsegregating here with vertebral fusion within Family KF2-01.

Gdf6 knockout mice express variable degrees of carpal and tarsalfusion; however, they do not manifest vertebral fusion. On theother hand, Gdf6/Gdf5 double knockout mice manifest bothcarpal/tarsal fusions and unique axial anomalies, including scoliosisand cellular defects within the developing intervertebral space, atthe future anatomical site of the disc [Settle et al., 2003]. Theseunique axial anomalies are consistent with discrete stripes ofGDF6 expression within the developing vertebral interspace aswell as suggesting a measure of Gdf redundancy in joint/discdevelopment. GDF5 and GDF6 are closely related and the level ofredundancy between the two molecules may extend further toexplain the variable degree of fusion (carpal and tarsal fusion) inGDF6 knockout mice and the variable degree of vertebral fusion(and carpal and tarsal fusion) within Family KF2-01. The extent ofvertebral fusion ranged from inconspicuous fusion bridges across asingle interspace (C2-3) in some individuals to others with largeblock fusions comprising six or more adjacent vertebrae. As such,the most plausible explanation for fusions (vertebral, carpal, and

FIGURE 5. Gdf6 knockdownphenotypes inXenopus laevis. Dorsal and lateral view of stage 47 tadpoles injectedwith 20 ngGdf6MOshowing range of ocular and axial abnormalities. Both10 ng and 20 ng ofGdf6 MOmRNA (RefSeqNM_001090364.1) gave a statis-tically signi¢cant yield inmorphant phenotypes (Po0.0074 andPo0.0001, respectively, one-sample t-test). A:Ocular defects alone(7%). Left to right panels show control, microphthalmia, coloboma, and anophthalmia. B:Ocular and axial defects (14%).Tadpolehas lateral kinked axis and left eye microphthalmia. C: Axial defects alone (39%). Phenotypic categories include: (i^ii) uninjectedcontrols; (iii^iv) kinked-dorsal/ventral and lateral, respectively; (v^vi) angled head/trunk in the dorsal ventral and lateral planes,respectively; and (vii^viii) curved axis-dorsal and lateral, respectively. In (v^viii) normal viscerawere displaced due to axial defects.

1024 HUMAN MUTATION 29(8),1017^1027,2008

tarsal) in this KFS family is the disruption of the long rangeregulation of GDF6 similar to camptomelic dysplasia, in whichintragenic and extragenic translocations disrupt the SOX9regulatory element�130 kb upstream of the gene itself [Wagneret al., 1994]. Functional studies in the mouse indicate the regiondownstream of GDF6 harbors GDF6 long-range regulatoryelements (D. Mortlock, personal communication) [Mortlocket al., 2003; Portnoy et al., 2005], and the 23 extremely wellconserved noncoding elements that are located downstream ofGDF6 on either side of the breakpoint (Fig. 3) may be part of theGDF6 regulatory/enhancer network perturbed by the breakpoint.

The identification of a missense mutation within the C-terminalhalf of the prodomain of the protein in another autosomaldominant KFS family provides further evidence for the role ofGDF6 in this disorder. The p.Ala249Glu mutation segregated withvertebral fusion in the three-generation Family KF2-03. Vertebralfusion patterns were always inclusive of the C2-3 fusion similar tothe pattern in Family KF2-01 and both families showed adiminishing frequency of more caudal fusions [Clarke et al.,1996, 1998]. Even though the type, range, and severity ofassociated anomalies may have varied between these two families,there was a precise positional correspondence evident between thefull familial range of skeletal anomalies (Table 1), the discretedevelopmental patterns of Gdf6 expression in mice (including thedevelopmental boundaries of the carpals and tarsals and theintervertebral spaces) [Mortlock et al., 2003; Portnoy et al., 2005],and Gdf6 knockout mouse phenotypes [Settle et al., 2003], whichsuggests that functional haploinsufficiency for GDF6 is onepossible mechanism of affect for the p.Ala249Glu mutation.

The recurrence of the p.Leu289Pro mutation, also in theprodomain, in two unrelated cases of the syndrome provides yetfurther compelling evidence for the role of GDF6 in KFS. Theprodomain regulates GDF6/BMP dimerization (GDF6 formshomodimers and heterodimers [Chang and Hemmati-Brivanlou,1999] with BMP2, which is also implicated in vertebraldevelopment [Chang and Hemmati-Brivanlou, 1999; Nakamuraet al., 2006]) and the functional importance of the leucine 289residue is supported by its extreme conservation both betweenspecies and between the prodomain of all three GDFs (which haveotherwise undergone considerable molecular divergence). Afterdimerization the prodomain is cleaved and GDF6 is secreted as anactive posttranslational modified, disulfide-bonded dimer. Suchdimerization is requisite for receptor binding activity and down-stream SMAD signaling and substitutions in the prodomain couldaffect either dimerization or downstream processing and cleavage(as observed with BMP4 [Goldman et al., 2006]) with potential forgain- or loss-of-function phenotypes [Cui et al., 2001]. Anonconserved substitution within the prodomain of GDF5 isknown to cause brachydactyly Type C [Everman et al., 2002] andhere we present the first evidence for similar deleterious mutationsin GDF6 associated with KFS skeletal defects.

The original GDF6 dosage protocols used to establish a linkbetween GDF6 and ocular defects in Xenopus [Hanel and Hensey,2006] were recapitulated here to expand the Xenopus phenotypeto include numerous axial defects in addition to and indepen-dently of eye defects. Even though GDF6 expression within thedeveloping eye and branchial arches and later within thedeveloping vertebral interspace were well documented (Table 1),axial defects went unrecognized in the prior investigation by Haneland Hensey [2006] due to their exclusive focus on eye formationearlier in development. Anterior axial defects were the mostprominent anomaly in Xenopus morphants and involved abnormalhead/trunk angles consistent with the anterior/cervical spinal

TABLE

2.Phen

otypes

Observed

inXen

opus

laev

isMorp

han

tsSco

redat

Dev

elopmen

talS

tage

50

Noey

ede

fect

Eye

defects:

microphthalmia/coloboma

Injectiontype

Axisnor

mal

(%)

Axishea

d/trunkan

gled

(%)

Axiskinke

d(%

)Axiscu

rved

(%)

Axisno

rmal

(%)

Axishea

d/trunkan

gled

(%)

Axiskinke

d(%

)AxisCurve

d(%

)Other

defect

(%)

n

Uninjected

53(100)

00

00

00

053

20ngControl

MO

84(92.3)

00

3(3.3)

2(2.2)

00

0Short

(noey

ede

fects)

(2.2)

91

10ngGDF6MO

54(70.1)

7(9.1)

4(5.2)

04(5.2)

3(3.9)

2(2.6)

0Short

(2.6);as

ymmetricmou

th(1.3)

7720

ngGDF6MO

74(49)

17(11)

11(7)

10(7)

10(7)

8(5)

5(3)

9(6)

Taildu

plic

ation(4);as

ymmetric

mou

th(1)

150

HUMANMUTATION 29(8),1017^1027,2008 1025

anomalies that distinguish KFS. The incomplete penetrance ofaxial, branchial, neural, and ocular anomalies observed here inmorphants parallels the variable phenotypes within and betweenthe familial and sporadic cases of KFS described here. Thisvariability suggests GDF6 thresholds may not only vary betweenindividuals and species but that GDF6 requirements for comple-tion of vertebral segmentation may vary between different regionsof the developing spine. Ophthalmological anomalies havebeen described in KFS [Gupte et al., 1992] and we are aware ofat least one patient who is unilaterally blind; however, it remainsto be seen if further mutation screening can identify a linkbetween GDF6 and ocular anomalies in KFS [Asai-Coakwellet al., 2007].

In conclusion, we have demonstrated disease-associated muta-tions at the GDF6 gene locus on chromosome 8 in both familialand sporadic cases of KFS (Types I–III), which help to explain boththe broad variability and diversity of KFS phenotypes. Theincomplete penetrance of carpal and tarsal fusions first observed inGDF6 knockout mice and again here in Family KF2-01 reflectwhat now appears to be a degree of redundancy and/or differencesin GDF6 thresholds required for normal joint development in boththe appendicular and axial skeleton. The role of GDF6 in neuraldevelopment, particularly as it may relate to mirror movementsand the issue of axonal guidance, provide interesting avenues forfurther research.

ACKNOWLEDGMENTS

We thank Dedee Murrell (Department of Dermatology StGeorge Hospital) for the provision of patient samples and Dr. ZhenLin for laboratory assistance. We thank Irene Smeaton, JamesBolan, Sarah Smith, and William Beckett for their assistance inthe UK sequencing initiative and Graeme Black for intellectualinput.We acknowledge the long-term interest of Martin F. Lavin(University of Queensland/Queensland Institute of MedicalResearch) in this project and his insight and comments duringthe preparation of this manuscript. M.T. was supported by theWellcome Trust (grant 061183).

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