Four novel mutations in the OFD1 (Cxorf5) gene in Finnish patients with oral-facial-digital syndrome 1 [5]

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LETTER TO JMG
Four novel mutations in the
OFD1 (Cxorf5)
gene in
Finnish patients with oral-facial-digital syndrome 1
A Rakkolainen, S Ala-Mello, P Kristo, A Orpana, I Järvelä
.............................................................................................................................
J Med Genet
2002;39:292–296
Oral-facial-digital syndrome type 1 (OFD1, MIM
311200) was first described by Papillon-Léage and
Psaume1in 1954 and further delineated in 1962 by
Gorlin and Psaume,2who called it orodigitofacial dysostosis. It
is a multiple congenital anomaly syndrome characterised by
malformations of the face, oral cavity, and hands and feet. The
facial dysmorphic features include hypertelorism, frontal
bossing, broad nasal bridge, hypoplasia of alar cartilage, and
transient milia. Oral cavity malformations include often
asymmetrical cleft of the palate (80%), small midline cleft of
the upper lip (45%), clefts of the tongue, hamartomatous
masses on the ventral surface of the tongue (70%), mucobuc-
cal fibrous bands, and dental abnormalities. Malformations of
the fingers are seen in 50-70% and toe malformations in 25%.
Central nervous system abnormalities, such as hydrocephalus,
porencephaly, and agenesis of the corpus callosum, with mild
mental retardation are seen in 40%.3In recent years, a kidney
disease closely resembling adult type polycystic kidney disease
has been shown to be one of the distinct features of this
syndrome.45
At least nine different forms of oral-facial-digital syn-
dromes have been described, type 1 being the most common
with a suggested incidence of 1:50 000 live births. OFD1 syn-
drome has dominant X linked inheritance with lethality in
males. However, a case of Klinefelter syndrome (XXY) with
OFD1 has been reported.6
By linkage analysis in two kindreds, the locus for OFD1 was
mapped to Xp22.3-22.2.7Recently, the gene for OFD1, Cxorf5,
was identified, and mutations of three familial and four
sporadic cases were identified by Ferrante et al.8Expression of
the gene was seen in all the tissues affected in the syndrome.
We report here the identification of four novel mutations in
the OFD1 gene together with the clinical findings in four
Finnish families, of which two are familial and two sporadic.
PATIENTS AND METHODS
Patients
The patients were ascertained from the Cleft Centre of the
Department of Plastic Surgery, Helsinki University Central
Hospital, where all patients with cleft lip and/or palate nation-
wide are treated. In addition, patients were ascertained from
the Department of Medical Genetics of The Family Federation
of Finland, which serves the whole country, and the Clinical
Genetics Unit of Helsinki University Central Hospital, which
serves the densely populated south of Finland in clinical
genetics. All the patients were examined (fig 1) and their files
and hospital records analysed by one of the authors (SA-M).
Mutation analysis
DNA extracted from peripheral EDTA blood of the patients
was screened for mutations in the OFD1 gene using primer
sequences kindly provided by Dr Brunella Franco from
Telethon Institute of Genetics and Medicine (TIGEM). PCR
amplifications of the samples were run through 35 cycles con-
sisting of 40 seconds at 94°C (denaturation), 40 seconds at 55
or 50°C (annealing), and one minute at 72°C (extension) with
Figure 1 The family pedigrees of the Finnish OFD1 families. Black symbols, affected; symbols with slashed lines, anamnestically affected.
I
1
2
3
II
1
32
III IV
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the final extension step of 5-10 minutes covering all 23 exons.
Sequencing of PCR products was performed using ABI
PRISM7 BigDye Terminator Cycle Sequencing Kit, Version 2.0
(Applied Biosystems, Foster City, CA, USA) in both directions
and analysed using an ABI PRISM7 3100 Genetic Analyzer
according to the manufacturer’s instructions. The presence of
a mutation was confirmed by minisequencing9of the DNA in
each family member. To exclude the presence of each of the
mutations in random subjects, DNA extracted from buffy coat
samples of 50 anonymous Finnish blood donors were analysed
by minisequencing.
Ethical approval for the study was obtained from the ethical
committee of Helsinki University Hospital and the Finnish
Red Cross Transfusion Service.
RNA analysis
RNA was isolated from heparin blood samples of the control
and the youngest patient from family I (fig 1) carrying the
intronic mutation IVS5-10T>G using the QIAamp RNA
Blood Mini kit (Qiagen, Hilden, Germany). This mutation
generates a putative novel splice site in exon 6. The mRNA
was reverse transcribed to cDNA using 1 µg of total RNA, 10
units of AMV reverse transcriptase (Promega M5101) in the
presence of 20 units of recombinant RNase inhibitor (RNasin,
Promega, N2511), and 25 nmol dNTPs. The reaction was
allowed to take place at 42°C for one hour, after which the
cDNA was diluted with 1.7 volumes of DNA-TE-Buffer (10
mmol/l Tris-HCl, pH 7.8, 1 mmol/l EDTA) and stored at −20°C.
cDNA synthesis was primed with the antisense primer
5′-ACTTGTCTGAGTTTCCATATTACAACTC-3′located in the
coding sequence of exon 6 of the OFD1 mRNA. For PCR two
sense primers were designed. The first one, 5′-
CATTAAAATCAACCCTACTTCCAGTCTC-3′, located in exon 4,
together with the reverse primer used in the reverse
transcription flanked the putative new splice site. The second
sense primer 5′-AGGATCTGATAAAGAAAATCAAAAAGGTTT
TTTAGGTTT-3′was designed to anneal exclusively over the
putative novel splice site to give a product only if this putative
new splice site was transcribed (fig 2).
RESULTS
We found four novel mutations in the OFD1 gene (table 1, fig
3) in two sporadic patients and in two families, both contain-
ing three patients with OFD1 syndrome (fig 1). The clinical
features of the patients shown in table 2 were characteristic of
OFD1 syndrome. In each case a novel mutation in the recently
discovered OFD1 gene was identified; two of them were
frameshifts, one was a missense mutation, and one was a
splice mutation.
In family I, the syndrome was diagnosed in three successive
generations (fig 1). The grandmother’s facial features were
typical of OFD1. She did not have cleft palate like her daugh-
ter and granddaughter. Instead, alveolar notching with miss-
ing teeth were seen. No abnormalities of the hands were seen.
At the age of 44 years, she had just undergone a kidney trans-
plant because of polycystic kidney disease. The kidney disease
had been discovered by chance on routine gynaecological
examination one year earlier and dialysis treatment was
started almost immediately after that. She was unwilling to
participate in genetic DNA studies. The daughter had small
hands and feet with brachydactyly of the fifth fingers. The
syndactyly of her fourth and fifth fingers of the left hand had
been operated on as a child. Renal ultrasonography was
performed at the age of 23, when the diagnosis of OFD1 was
confirmed. Multiple cysts were seen in the right kidney,but no
signs of renal failure in the laboratory examinations was
found. The granddaughter, aged 1.5 years, has developed nor-
mally. In the extremities, there was only mild clinodactyly of
the fifth fingers. The cleft palate was asymmetrical. Alveolar
notching, suggesting tooth aplasia, and mucobuccal fibrous
bands were seen. No signs of retardation were detected in this
family. We found a T>G change in intron 5 of the OFD1 gene
in the daughter and the granddaughter. The mutation is
located 10 nucleotides before the starting nucleotide of exon 6
(fig 3) where it creates a novel splice acceptor site (and adds
three novel amino acids to the 5′end of exon 6) resulting in an
alternative splicing of mRNA. This was confirmed by the RNA
studies described in the Methods section (fig 4).
In family II (fig 1), the mother and her two daughters were
clinically examined and their facial features and other signs
were typical of OFD1 syndrome (table 2). All three patients
studied had midline pseudocleft of the upper lip, but no
operations had been performed. The tongues of the mother
and the older daughter were bilobulated and the younger
daughter had multiple lobules in her tongue. No-one in this
family had had problems with kidney function and no ultra-
sonographic examinations of the kidneys were performed. At
the age of 42 years, the mother was diagnosed with
hyperthyreosis, which was treated with radioactive iodine.
The younger daughter had been operated on at the age of 1
year because of a medially located, supernumerary distal
phalanx in the right hallux. The left leg grew 3 cm longer
than the right leg and at the age of 13 years an orthopaedic
operation was performed. The left breast has grown bigger
than the right with mastopathic changes. Her mental devel-
opment has been mildly delayed and she attended a special
school. In the older daughter, vaginal bleeding started at the
age of 3 months. After investigations, hormonal medication
was given for precocious puberty. Epileptic seizures began at
the age of 2
1
⁄
2
years. Repeated CT scan of the brain showed a
hypothalamic hamartoma, which was thought to be the rea-
son for the precocious puberty through excretion of hypotha-
lamic hormones. She had short stature with a final height of
1.45 m (−3.5 SD) and small hands and feet. The fourth meta-
tarsals were short, especially in the right foot. She attended a
Figure 2 Diagram of detection of the transcript showing the
abnormal splicing caused by the IV5-10T>G mutation in exon 6 of
the
OFD1
gene.
Exon 5
Exon 5 Exon 6Mut
Exon 5 Exon 6
RT-PCR product (+)
RT-PCR product (–)WT
Exon 6
IVS5-10T>G Table 1 Mutations in patients with OFD1
Family
(case*) Location Nucleotide change† Effect on protein
I (F) Intron 5 IVS5-10T>G Abnormal splicing
II (F) Exon 16 1887-1888insAT Frameshift
III (S) Exon 3 235G>A A79T†
IV (S) Exon 13 1409delA Frameshift
*F=familiar, S=sporadic.
†Mutation description is according to Antonarakis
et al
12 with the
cDNA sequence of
OFD1
used as the reference and with the ATG
translation initiation codon denoted as nucleotide +1.
Letters 293
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special school for handicapped children because of moderate
mental retardation and received medication for psychiatric
symptoms for a couple of years. In this family, an insertion of
AT between nucleotides 1887 and 1888 in exon 16 was
detected in all three family members (fig 3). This creates a
frameshift resulting in a premature stop codon (TAG) at
amino acid position 666 of the OFD1 gene.
In family III, the only patient studied had syndactyly of the
fourth and fifth fingers of the left hand that had been oper-
ated on at the ages of 5 and 11 years. On ultrasonographic
Table 2 Clinical features of the patients with OFD1
I.3 I.2 IV III II.3 II.2 II.1
Age (years) 1.5 23 0.5 30 19 25 50
Clinical findings
Facial
Midfacial flattening ++++−−+
Alar hypoplasia +++++++
Dystopia canthorum + + + −+−+
Skin milia + −+−−−−
Oral
Thinupperlip +++++++
Cleft palate + + −−−−−
Midline pseudocleft of upper lip −−−−+++
Alveolar notching ++++−++
Toothaplasia NA+NA++++
Lobulated tongue +++++++
Tongue hamartoma + + + −++−
Multiple frenula ++++−+−
Cerebral
Mental retardation −−−−++−
Renal
Polycystic kidneys ND + ND + ND ND ND
Extremities +++++++
NA=not yet available, ND=not done.
Figure 3 Sequencing chromatograms showing the four
OFD1
mutations in the Finnish patients.
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examination, numerous small cysts were detected in both
kidneys at the age of 29 years. Functional studies of the
kidneys were normal. In this patient a missense mutation
G>A at nucleotide 235 in exon 3 was identified (table 1, fig
3). This transversion leads to a change of a non-polar amino
acid alanine (A) to an uncharged polar amino acid threonine
(T). We analysed DNA samples from both parents by minise-
quencing and no abnormalities were found, indicating that
this is a de novo mutation.
In family IV, the index case was first examined at the age of
6 months. The first diagnostic signs were a prominent metopic
ridge and a soft nodule (about 0.5 cm in diameter) medially in
the right hallux. Psychomotor development has proceeded
within normal limits. In this patient, a deletion of A at nucleo-
tide 1409 in exon 13 leading to a frameshift was identified.
This mutation results in a premature stop codon (TAG) at
position 472. DNA from both parents was analysed and no
mutations were found.
None of the four mutations was identified in the DNA of 50
anonymous Finnish blood donors screened by minisequenc-
ing.
RNA
The results of the RT-PCR experiments (fig 4) show that in
both the patient and the control sample the products
generated by RT-PCR amplifying the area flanking the putative
novel splice site are of similar size, indicating that the normal
sized mRNA could be found in both samples. However, the
splice site specific RT-PCR resulted in the identification of the
product only in the patient’s sample. This indicates that the
intronic nucleotide change T >G residing 10 nucleotides from
the splice acceptor site of exon 6 generates a false splice site
and so is most likely the cause of the disease in this patient.
DISCUSSION
Eight OFD1 patients have been diagnosed in Finland, consist-
ing of a population of about 5 million, during the last 20 years.
In all of them, a mutation in the recently identified OFD1
(Cxorf5) gene was found. Two of them were nonsense, one
missense, and one splice mutation. The clinical features were
characteristic in every patient. Interestingly, one of our
patients had short fourth metatarsals, similar to a patient
described by Ferrante et al.8Mild or moderate mental retarda-
tion was seen in one of the families with the two daughters
with learning difficulties.
Renal involvement in OFD1 cases may be as high as 50%.10
In three out of eight Finnish patients, polycystic kidney
disease was present, and one of them received a new kidney at
the age of 44 years. The mutations that were associated with
polycystic kidney disease in the Finnish patients were the
splice mutation in intron 5 and a missense mutation G>A at
nucleotide 235 in exon 3. In the original report by Ferrante et
al,8polycystic kidney disease was also associated with
mutations in exon 3 but also in intron 4. Polycystic kidney
disease usually manifests in adulthood, so two of our patients
are too young to be able to draw any conclusions about kidney
disease.
When analysing the phenotype-genotype correlation con-
cerning mental retardation associated with this syndrome,
mild to moderate mental retardation or learning difficulties
were reported with mutations in exons 3, 13, and 16, and
intron 4 in the original study.8In this study, only the
frameshift mutation in exon 16 was associated with learning
difficulties in two out of three members of the same family.
Further studies are needed to know whether certain
mutations are more frequently associated with kidney disease
or mental retardation, the findings that are important in
genetic counselling when predicting the outcome of the
disease.
The OFD1 gene contains 23 coding exons (GenBank acces-
sion numbers Y15164 and Y16355) with unknown function.11
Interestingly, three of the mutations found in this study are
located in the same exons 3, 13, and 16 as the mutations
reported in the original study by Ferrante et al,8suggesting
that these exons might represent regions for mutational hot
spots. Functional studies of both the wild type OFD1 gene and
the mutants are needed to understand the disease mechanism
underlying OFD1.
In conclusion, we report here the identification of four novel
mutations in the OFD1 gene in seven Finnish patients with
oral-facial-digital syndrome type I. Our results confirm the
causative role of the OFD1 gene in the pathogenesis of this
syndrome.
ACKNOWLEDGEMENTS
We are grateful to the patients and their families for their
participation in this study. We thank Sirkka Elfving and Eino
Puhakainen for encouragement during this study and the personnel
Figure 4 The RT-PCR-products covering exons 4-6 are normal in
both control and patient samples (on the left). The intronic nucleotide
change IVS5-10T>G results in an abnormally spliced product in the
patient sample (RT(+)) compared to the normal sample (RT(+)). RT(−)
samples are the control samples with no cDNA.
Φ X174
Hae
III
Control, RT(+)
Control, RT(–)
Patient, RT(+)
Patient, RT(–)
Ex4 – Ex6
Φ X174
Hae
III
Control, RT(–)
Patient, RT(+)
Patient, RT(–)
Control, RT(+)
Splice site mutation
+Oral-facial-digital syndrome type 1 (OFD1) is an X linked
dominant disorder characterised by malformations in the
face, oral cavity, and digits with a wide phenotypic vari-
ation. Recently, mutations in the
OFD1
gene (
Cxorf5
)at
Xp22 were found to underlie OFD1. We report here the
identification of four novel mutations in the
OFD1
gene
in the Finnish families, two of which are familial and two
sporadic.
+In the familial cases a splice mutation T>G in intron 5 in
the mother and her daughter was identified resulting in
an abnormal splicing, and in the second family a
nonsense mutation 1887-1888insAT in exon 16 was
detected in the mother and her two daughters. Analysis
of the sporadic cases showed a missense mutation
235G>A in exon 3 and a single nucleotide deletion
1409delA leading to a nonsense mutation in exon 13.
Three of the mutations in this study were located in the
same exons as in the original study.
+Our study confirms the causative role of the
OFD1
gene
in the pathogenesis of oral-facial-digital syndrome type
1.
Letters 295
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of the Laboratory of Molecular Genetics for technical help. Financial
support from Helsinki University Hospital Research Funding is
acknowledged.
.....................
Authors’ affiliations
A Rakkolainen, A Orpana, Department of Clinical Chemistry,
University of Helsinki, Helsinki, Finland
A Rakkolainen, S Ala-Mello, P Kristo, A Orpana, I Järvelä,
Department of Medical Genetics, University of Helsinki and
HUCH-Laboratory Diagnostics, Helsinki, Finland
S Ala-Mello, Clinical Genetics Unit, HUCH-Laboratory Diagnostics,
Helsinki, Finland
Correspondence to: Dr I Järvelä, HUCH-Laboratory Diagnostics,
Laboratory of Molecular Genetics, Haartmanink 2, 00290 Helsinki,
Finland; irma.jarvela@hus.fi
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ECHO.................................................................................................................
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both susceptibility to multiple sclerosis and its outcome.
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multiple sclerosis from 166 families and reanalysed the data for the new total of 262 pairs from 250
families. As before, they looked for concordance in clinical variables in each pair of siblings for course of
disease, presenting symptoms, age and year of onset—and this time also included measures of disability,
disease progression, and handicap. The data were adjusted for confounding factors associated with
analysis of sibling pairs and were analysed with statistical techniques that can include potentially con-
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ease will converge such that each sibling in a pair will eventually have similar disability and handicap
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m
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