Expanding the molecular basis and phenotypic
spectrum of X-linked Joubert syndrome associated
with OFD1 mutations
Michael Field*,1, Ingrid E Scheffer2,3,4, Deepak Gill5, Meredith Wilson6, Louise Christie1, Marie Shaw7,
Alison Gardner7, Georgie Glubb3, Lynne Hobson7, Mark Corbett7, Kathryn Friend7, Saffron Willis-Owen7,8
and Jozef Gecz*,7,9,10
Using a combination of linkage mapping and massively parallel sequencing of the X-chromosome exome, we identified an 18-bp
deletion in exon 8 of the oral-facial-digital syndrome type 1 (OFD1) gene in a family with X-linked Joubert syndrome (JBTS10).
The deletion results in an in-frame deletion of six amino acids. New features not noted in the two previously reported cases
of X-linked Joubert syndrome include the presence of polycystic kidney disease, polymicrogyria and hydrocephalus. Our study
further underlines the power of genetic mapping combined with massively parallel sequencing as a powerful tool for novel
disease gene and mutation discovery.
European Journal of Human Genetics (2012) 20, 806–809; doi:10.1038/ejhg.2012.9; published online 22 February 2012
Keywords: OFD1; X-linked Joubert; X-linked intellectual disability; massively parallel sequencing
The oral-facial-digital syndromes (OFDSs) are a group of nine dis-
orders whose features include midline oral clefts (lip, palate and
tongue), nodules (hamartomas) on the tongue, and digital abnormal-
ities, including brachydactyly, syndactyly and polydactyly. Both poly-
cystic kidney disease and central nervous system anomalies including
hydrocephalus and cerebellar anomalies are reported in some cases.
OFDS type 1 (OFD1) is the X-linked dominant form associated with
male lethality and the presence of polycystic kidneys. Mutations in the
OFD1 gene (MIM 311200) were first identified in 2001.1
Joubert syndrome is characterized by the presence of cerebellar
vermis hypoplasia, hypotonia, ataxia, oculomotor apraxia and ir-
regular respiration with tachypnea followed by apnea in the neonatal
period. Abnormalities may be present in other organs including the
kidney, liver and eye. A key neuro-imaging finding is the molar tooth
sign (MTS) reflecting a deepening of the interpeduncular fossa at the
level of the isthmus and upper pons, elongation, thickening and mal-
orientation of the superior cerebellar peduncles and cerebellar vermis
hypoplasia. The MTS is not absolutely pathognomonic of this disorder
and may be seen in other conditions including OFD type VI.2With
advances in our molecular understanding of these conditions, it has
become apparent that some of these disorders are allelic.
Coene et al3reported mutations in the OFD1 gene in a family with
X-linked Joubert syndrome (JBTS10) (MIM 300804) and in an
isolated male with Joubert syndrome. Clinical features included severe
intellectual disability with absent speech and severe motor impair-
ment, MTS on imaging, juvenile retinitis pigmentosa in the familial
case and bilateral polydactyly. Females were unaffected. For the first
time, the OFDS phenotype and JS phenotype was shown to be allelic.
More recently, mutations in TMEM216 (MIM 613277) have been
identified in patients with JS and OFD type VI, again illustrating the
overlap of these phenotypes.4We report a new family with X-linked
Joubert syndrome in which linkage mapping combined with massively
parallel sequencing of the X-chromosome exome revealed an in-frame
18-bp deletion within exon 8 of the OFD1 gene. In addition, we
extend the phenotype associated with mutations in OFD1.
SUBJECTS AND METHODS
Two distant male maternal cousins V-3 and V-9 now aged 7 and 11 years,
respectively, who were related through six female relatives (Figure 1), were
independently identified to have common neuroradiology and developmental
features. V-3 was noted on second trimester antenatal morphology scans to have
at term by a breech normal vaginal delivery with a birth weight of 3520g
(50th centile) and head circumference of 39.5cm (497th centile). Motor mile-
stones were delayed, sitting independently at 12 months and walking independently
at 5 years of age. He had no focal neurology signs, nystagmus or intention tremor.
He had hypermetropia and an intermittent esotropia requiring visual correction.
There was a large discrepancy between his receptive and expressive language skills.
At 6 years of age, he was non-verbal, but was able to sign, use an assisted
communication device and read some age appropriate material.
Received 10 August 2011; revised 24 November 2011; accepted 26 December 2011; published online 22 February 2012
1Genetics of Learning Disability Service, Newcastle, New South Wales, Australia;2Florey Neurosciences Institute, Melbourne, Victoria, Australia;3Department of Medicine,
University of Melbourne, Austin Health, Melbourne, Victoria, Australia;4Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Melbourne, Victoria,
Australia;5TY Nelson Department of Neurology, The Children’s Hospital at Westmead, Westmead, New South Wales, Australia;6Department of Clinical Genetics, Children’s
Hospital at Westmead, Westmead, New South Wales, Australia;7SA Pathology at the Women’s and Children’s Hospital, Neurogenetics, North Adelaide, South Australia, Australia;
8Imperial College, London, UK;9School of Paediatrics and Reproductive Health and School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South
Australia, Australia;10Women’s and Children’s Health Research Institute, North Adelaide, South Australia, Australia
*Correspondence: Dr M Field, Genetics of Learning Disability Service, PO Box 84, Waratah, New South Wales 2298, Australia. Tel: +61 2 4985 3136; Fax: +61 2 4985 3105;
or Dr J Gecz, SA Pathology at the Women’s and Children’s Hospital, Neurogenetics, 72 King William Road, North Adelaide, South Australia 5006, Australia. Tel: +61 8 8161 6339;
Fax: +61 8 8161 7342; E-mail: email@example.com
European Journal of Human Genetics (2012) 20, 806–809
& 2012 Macmillan Publishers Limited All rights reserved 1018-4813/12
V-9 was delivered by cesarean section at 39 weeks, with a birth weight of
3485g (50th centile) and head circumference of 36cm (75th centile). He was
macrocephalic by 4 months, and had asymmetric spasticity in his upper limbs
worse on the left side at 6 months. He sat independently at 33 months and
walked with ankle-foot orthoses after 5 years. He had single words at 20
months and at 10 years of age spoke in short phrases with significant dysarthria
and dyspraxia. Recent developmental assessments showed borderline/low non-
verbal cognitive skills. EEG studies persistently showed epileptiform activity
with a right centrotemporal focus. He had febrile seizures from 2.5 years, an
afebrile focal dyscognitive seizure at 4.5 years followed by infrequent general-
ized tonic-clonic seizures treated with carbamazepine.
Both boys had macrocephaly (497th centile) with frontal bossing, V-9 has
mild down- sloping palpebral fissures with epicanthic folds and V-3 deep-set
eyes with infra-orbital creases (Figure 2a). Neither had digital anomalies or oral
abnormalities including dental abnormalities, clefting or frenulae. There was no
evidence of retinitis pigmentosa on formal ocular examination in either boy at
5 years. A renal ultrasound in V-3 identified renal cystic disease at 5 years of
age. His renal function has subsequently deteriorated, and he is awaiting
transplantation. V-9 had increased echogenicity of his kidneys at 6.5 years
without obvious cysts or renal impairment. Neither boy had cardiac abnorm-
alities Neuro-imaging showed a number of similarities including horizontal
cerebellar peduncles, an enlarged cisterna magna and a MTS (Figure 2b). V-9
also had extensive polymicrogyria of the right frontal and temporal lobes
(Figures 2c and d).
The family history included two further affected males whose cerebral
abnormalities may have been manifestations of the X-linked Joubert syndrome.
A male sibling V-2 was identified on antenatal scan and at postmortem to have
significant hydrocephalus at 19 weeks gestation. Antenatal scans suggested a
posterior fossa abnormality, but macroscopically this could not be confirmed at
postmortem. A maternal uncle IV-3 died after birth of complications relating to
hydrocephalus and a cyanotic congenital heart anomaly.
Based on the pedigree structure, sequencing of a number of candidate
genes on the X chromosome including OPHN1, L1CAM and AP1S2
was performed, without identification of a causative mutation. When
we began our study OFD1 had not been identified as the cause
of X-linked Joubert syndrome. Subsequent X chromosome linkage
analysis was carried out (ABI Prism (Melbourne, Victoria, Australia)
Linkage Mapping set HD) on available samples, including three
obligate carriers, two affected and four unaffected males. Maximum
two point LOD scores of 2.27 at y¼0 were achieved for DXS987 and
DXS7593, with recombinant events detected at DXS7108 and
chrX:10052226–27452338; linkage data not shown) (Figure 3a).
In the absence of an obvious candidate gene within the linkage
interval, massively parallel sequencing of the X-chromosome exome
(Agilent, Melbourne, Victoria, Australia) was performed (Illumina GA
II; Geneworks, Adelaide, South Australia, Australia) on the proband’s
(V-3) whole blood isolated DNA (see Supplementary Information). A
mean of 84.3-fold coverage was achieved in bait regions (median 78),
and 86.9% of bait bases were captured at a coverage depth Z10.
A total of 23 discrepant sites met filtration criteria, all of which
demonstrated the only or highest quality alignments to chromosome
X in UCSC blat searches. Of these 23 sites, 4 yielded missense sequence
changes (Figure 3b). These changes included p.V328I in MAGEB10,
p.F101C in SSX1, p.R47W in TIMM17B and p.L15Q in TREX2
(Supplementary Table 1). None of these changes localized either
within or in the proximity of our family’s linkage interval. As such
the stringency of the filtration criteria was relaxed to include sequence
discrepancies present at lower frequencies (Z30% reads). This
approach generated a total of 135 sites for investigation, each of
which was examined manually in Consed. Among these 135 sites, 1
was found to be consistent with an 18-bp deletion in the eighth exon
of the OFD1 gene (Figures 3c and d). This previously unreported
deletion results in a six amino-acid deletion without a frameshift.
The deletion was confirmed by Sanger sequencing (Supplementary
Figure 1) and segregated completely with the phenotype (Figure 1).
The phenotypic spectrum associated with OFD1 mutations has been
recently extended. The spectrum includes a syndrome including
macrocephaly, severe mental retardation, ciliary dyskinesia leading
to recurrent respiratory infections and in some cases polydactyly5(also
referred to as Simpson–Golabi–Behmel type 2), an X-linked Joubert
phenotype and the classical OFD1 pattern with male lethality.3This
case confirms the reported association of specific OFD1 mutations
with X-linked Joubert syndrome. Unlike previous reported cases of
males with an OFD1 mutation, our patients show relatively
well-preserved non-verbal cognitive abilities. This does not seem
Figure 1 Family pedigree. ASD, autism spectrum disorder. *Indicates DNA available for linkage analysis. Genotype data are given as mutation and/or
OFD1 deletion in X-linked Joubert
M Field et al
European Journal of Human Genetics
uncommon in patients with Joubert syndrome, with their receptive
language skills often being advanced and gross motor skills often being
the weakest domain.6
The extended anomalies seen in this family include polycystic
kidney disease, hydrocephalus, polymicrogyria and possibly congenital
heart disease. Hydrocephalus is reported in a minority of females with
OFD1, as is cerebellar hypoplasia and Dandy Walker malformation,7,8
but the spectrum of anomalies can extend to cerebral dysgenesis with
evidence of a severe neuronal migration abnormality in one female
fetus.9A recent review suggests that the presence of polycystic kidneys
is positively associated with structural neurological abnormalities in
OFD1 patients.7Functionally, in both the mouse and zebrafish more
complete loss of OFD1 function has been shown to be associated with
abnormal ciliary function, with an extreme manifestation being
hydrocephalus and laterality defects including cardiac anomalies.10,11
The congenital heart lesion and hydrocephalus reported in IV-3 is
compatible with the animal model phenotype for OFD1 loss of
It has been suggested that the phenotype associated with OFD1
mutations is dependent on the location of the mutation and how it
impacts on nuclear localization and binding with Lebercilin.3Until
this report, it was thought that mutations proximal to exon 17 would
always be associated with male lethality or females with an OFD1
phenotype. The mutations identified in patients with OFD1 are
predominantly truncating, with the few reported missense mutations
clustering in a LisH motif in exon 3.8,12To our knowledge, mutations
in exon 8 have all been splice site or frameshift. Our family with a six
amino-acid in-frame deletion in exon 8 demonstrates that the
relationship between location and phenotype is more complex than
initially hypothesized. We suspect this in-frame deletion in a male
must reduce OFD1 expression, to a lower level than in a female who is
heterozygous for a nonsense mutation, but is not associated with male
This family is the third family with an X-linked Joubert
phenotype to be reported with an OFD1 mutation. The case
extends the spectrum associated with OFD1 mutations and illustrates
that a proximally placed mutation that is non-truncating will not
always result in the classical OFD1 phenotype. The small linkage
interval allowed us to rigorously analyze next-generation sequencing
data and by softening our search criteria, to identify the 18-bp deletion,
which could otherwise be overlooked. With the ongoing advancement of
the massively parallel sequencing technologies and analysis tools13
and paired-end sequencing in particular, such deletions will be a lot
easier to identify.
Figure 2 Clinical data. (a) Photograph of V-3 – note prominent forehead and macrocephaly. (b) MRI scan V-9. Axial T2 image showing the MTS with a
small right cerebral peduncle (RCP). (c) MRI scan V-9. Axial T2 image showing extensive right hemispheric polymicrogyria with sulcation abnormalities.
(d) MRI scan V-9. T1 reformatted sagittal image through the lateral right hemisphere showing extensive polymicrogyria.
OFD1 deletion in X-linked Joubert
M Field et al
European Journal of Human Genetics
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Supplementary Information accompanies the paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)
Figure 3 Identification of the OFD1 gene 18-bp deletion. (a) The disease gene in this family has been mapped to an Xp22.2–Xp21.3 interval using
traditional linkage mapping. (b) Following X-chromosome exome capture and massively parallel sequencing the variants (referred to as highly discrepant,
or HD sites) were filtered independently of the linkage interval. (c) Relaxing the stringency of filtering lead us to the identification of a region in OFD1, which
upon further adjustment revealed an 18-bp deletion within exon 8 of OFD1. (d) This deletion was subsequently predicted to result in an in-frame deletion
of six amino acids without the introduction of a missense change.
OFD1 deletion in X-linked Joubert
M Field et al
European Journal of Human Genetics